Background & Aims

Chronic low back pain (CLBP) is highly prevalent (~8% globally) and is the leading cause of years lived with disability worldwide.1 Previous work shows that peripheral immune cells, such as neutrophils, monocytes, and T cells, have a role in the chronicity of pain and are under circadian control. Likewise, CLBP has been shown to vary in intensity throughout the day; however, the mechanisms underlying this variability remain unclear at the molecular and cellular levels. This study aims to investigate how CLBP pain rhythmicity affects immune cells and the transcriptomic changes underlying their responses.

Methods

Pain rhythmicity phenotypes (constant-low, constant-high, rhythmic? and mixed) were characterized in 74 participants with CLBP. Peripheral blood samples (n=116) were collected at 8:00 and 20:00 for bulk RNA-sequencing. Reads were processed using FastQC/MultiQC, Hisat2, and StringTie. Normalization and outlier detection applied edgeR and arrayQualityMetrics. Transcripts with median absolute deviations ? the 70th quantile (n=101,0350) were kept for analysis. We identified differentially expressed transcripts (DETs) between the rhythmic? and other phenotypes using edgeR. Unsupervised network analysis identified transcript clusters associated with pain rhythmicity?. Gprofiler2 ran pathway analysis on significant transcripts. Using the PainOMICs LBP cohort2 to replicate results, DESeq2 assessed whether a gene’s trajectory between two visits differs between opioid users vs non-users. Enrichment analysis (fgsea) focused on Gene Ontology’s biological processes (GO:BP) about cell activation.

Results

We identified 40 to 170 DETs between the rhythmic? and other pain phenotypes (PBonferroni<0.05). Pathway analyses determined significant enrichment of immune cell signaling and neutrophil degranulation pathways (Padj.<0.05). Network analysis clustered transcripts into 82 and 77 clusters of co-expressed transcripts in the day and night networks, respectively. The rhythmic? phenotype was associated with 3 day clusters and 5 night clusters (P<0.05). Further, the neutrophil degranulation pathway was enriched in a night cluster negatively associated with rhythmicity? (Padj.<10e-57). Moreover, among all cell types of hematopoietic origin whose activation pathway were documented in GO:BP, only neutrophils showed significant activation (FDR<0.01). Finally, the neutrophil degranulation pathway’s genes were over-expressed over time in opioid users versus non-users (enrichment score +0.31, P=2e-6).

Conclusions

In this study, our results suggest that neutrophil activation may differentiate rhythmic?, non-opioid users from other pain phenotypes. Specifically, there is less neutrophil degranulation amongst patients with a rhythm of CLBP intensity and amongst those who do not consume opioids. These findings support previous evidence that neutrophils play a role in chronic pain and are under circadian control. Indeed, neutrophils have been suggested to relieve pain by secreting opioid peptides at the site of inflammation and inhibiting T-cell responses. Neutrophils have also been shown to have circadian rhythms in their gene expression and trafficking in the naïve and injured states. However, the role of neutrophil activation in the rhythmicity of pain intensity has not previously been described. Hence, circadian rhythms of pain and neutrophil activation may guide novel interventions for individuals with chronic low back pain.

References

1.GBD 2021 Low Back Pain Collaborators. Global, regional, and national burden of low back pain, 1990-2020, its attributable risk factors, and projections to 2050: a systematic analysis of the Global Burden of Disease Study 2021. Lancet Rheumatol. 2023;5(6):e316-e329. Published 2023 May 22. doi:10.1016/S2665-9913(23)00098-X
2.Parisien M, Lima LV, Dagostino C, et al. Acute inflammatory response via neutrophil activation protects against the development of chronic pain. Sci Transl Med. 2022;14(644):eabj9954. doi:10.1126/scitranslmed.abj9954

Presenting Author

Amanda Zacharias

Poster Authors

Amanda Zacharias

BSc

Queen's University

Lead Author

E-mail

Marc Parisien

McGill University

Lead Author

E-mail

Hailey Gowdy

Queen's University

Lead Author

E-mail

Mitra Knezic

Queen's University

Lead Author

E-mail

Doriana Taccardi

Queen's University

Lead Author

E-mail

Etienne Bisson

PhD

Kingston Health Sciences Centre

Lead Author

E-mail

Daenis Camiré

MD

Kingston Health Sciences Centre

Lead Author

E-mail

Elizabeth Brown

RN

Kingston Health Sciences Centre

Lead Author

E-mail

Tracey Stevenson

RN

Kingston Health Sciences Centre

Lead Author

E-mail

Ana Constantin

Queen's University

Lead Author

E-mail

Jesse Joynt

Queen's University

Lead Author

E-mail

Rosemary Wilson

PhD

Queen's University

Lead Author

E-mail

Lesley Norris Singer

PT

McGill University

Lead Author

E-mail

Manon Choinière

CHUM - Centre de Recherche (CRCHUM)

Lead Author

E-mail

Gabrielle Pagé

University of Montreal

Lead Author

E-mail

Luda Diatchenko

McGill University

Lead Author

E-mail

Qingling Duan

PhD

Queen's University

Lead Author

E-mail

Nader Ghasemlou

Queen's University

Lead Author

E-mail

Topics

  • Specific Pain Conditions/Pain in Specific Populations: Low Back Pain