Biomarker signatures for sickle cell pain Open Access

In this issue of Blood Advances, Mucalo Katunaric et al¹ present a transcriptional biomarker signature associated with the frequency of pain in individuals with sickle cell disease (SCD). They identified 10 top hub genes associated with inflammation and pain, which included adhesion molecules, cytokines/chemokines, and complement receptors. Having objective biologic biomarkers of pain in SCD is a critical unmet need. The authors adopted a novel approach to identify the transcriptional signature of pain in individuals with SCD (see figure). Their unique approach involved using cryopreserved peripheral blood mononuclear cells (PBMCs) from a single healthy donor and coculturing it with the plasma from each participant with SCD, an approach that previously has been used successfully in studies of diabetes, cystic fibrosis, and inflammatory bowel disease.

The plasma used was from individuals with SCD who were aged 4 to 21 years from a previous multisite randomized control trial named Magnesium for Children in Crisis (MAGiC; ClinicalTrials.gov identifier: NCT01197417),² and biobanked plasma from black individuals aged >7 years without SCD or any pain condition were used as healthy controls. The MAGiC cohort included patients with homozygous (HbSS) and patients with HbSβ⁰-thalassemia who required hospitalization after failing management of pain in the emergency department. The robust final analysis was conducted on 126 SCD plasma samples. Weighted gene coexpression network analysis (WCGNA) for the identification of diagnostic and prognostic biomarkers; gene ontology using Database for Annotation, Visualization and Integrated Discovery; and Search Tool for the Retrieval of Interacting Genes (STRING) for the protein-protein interaction (PPI) network were used. Correlations were drawn between WCGNA-derived gene modules and acute care visits in the previous 3 years, which identified 524 transcripts that differed between the SCD cohort and the cohort without SCD. The authors identified 10 hub genes and interactions (based on PPI/STRING), which included proinflammatory cytokines and chemokines, adhesion molecules, and the complement/integrin system known to be associated with vascular sickle cell pathobiology and/or neutrophil recruitment. The constituents of plasma that induce transcriptional modulation in PBMCs remain to be understood.

In general, the top hub cytokines/chemokines are known to evoke inflammatory signaling via the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway, which leads to transcriptional activation and a proinflammatory response.³ Indeed, a highly phosphorylated JAK/STAT pathway has been observed in the neutrophil lysates and proteosome of individuals with homogenous SCD.⁴ Many JAK/STAT pathway inhibitors are in clinical use but have not been tested in SCD.³ In preclinical studies of humanized transgenic HbSS-BERK sickle mice, spinal JAK/STAT signaling was associated with central sensitization.⁵ In NY1DD sickle mice, JAK/STAT signaling was evoked by morphine in the retinal endothelium, which led to increased transcription and expression of interleukin-6 (IL-6).⁶ In the retinal endothelial study, only IL-6 was analyzed as a representative of the regulation of proinflammatory cytokines induced by morphine. It is plausible that some individuals with SCD in this study have been on pain management medications, including opioids, which could have led to increased expression of cytokines in the plasma and, in turn, may have activated the JAK/STAT pathway in the cells and may have led to the increased transcription and expression of proinflammatory molecules such as those observed by Mucalo Katunaric et al. In addition, proinflammatory pathways of the NLR family pyrin domain containing 3 inflammasome and nuclear factor κ-light-chain-enhancer of activated B cells are activated by many cytokines/chemokines, which would further enhance the inflammatory response. Together, the cytokine/chemokine expression induced by sickle plasma in this study is highly insightful for the development of targeted therapies for pain.

Notably, adhesion molecules, including intercellular adhesion molecule 1 (ICAM1) and/or complement activation, have been shown to contribute to vaso-occlusion. The complement activation fragments Bb and C5a, together with ICAM1, were increased following cold-induced vaso-occlusion in HbSS Townes sickle mice.⁷ In addition to the complement system, increased C-C motif ligand 2 (CCL2)/monocyte chemoattractant protein 1 also contributes to cold hyperalgesia in sickle mice.⁸ In addition, endothelial integrins mediate the adhesion of sickle red blood cells to the endothelium via adhesion molecules, such as thrombospondin.⁹ Preclinical studies showed that the C-C chemokine motif receptor 5 (CCR5) may underly neuronal injury, sprouting, and dendritic spine injury in the brain following a traumatic brain injury or stroke.¹⁰ It is likely that complement activation and many cytokines/chemokines (CCL2 and others) and their receptors, such as CCR5, play a causative role in the complex pain phenotype in SCD exhibited in the form of inflammatory, nociceptive, and neuropathic pain. This study’s outcomes also validate preclinical data from transgenic sickle mice.

It is intriguing to know if the plasma and PBMCs from the same individual would provide more personalized information. However, the evidence of the top hub transcripts with evidence for inciting inflammation and neutrophil recruitment, as well as pronociceptive activity and association with pain frequency provide confidence that this approach can be advanced clinically and in reverse translation studies. Many other cells, such as basophils, may be induced in vivo, and this approach may be extended to examining the impact on a cocktail of cells, which may provide a more comprehensive readout. It is also likely that the molecular signatures may change with disease progression. Authors have recognized the need for a longitudinal analysis. Age and sex did not contribute to the measured variance. It is likely that a larger number of participants may be required to distinguish between sexes and age dependence. Although the authors suggest that these biomarkers are diagnostic and prognostic, it is likely that this approach can be applied as biomarkers to determine the response to therapy. The simplicity of their approach, which only requires a small amount of blood, is highly conducive to repeated longitudinal analysis. However, it needs to be determined if these observations are a cause or a consequence.

In conclusion, Mucalo Katunaric et al provide a foundation for the advancement of biomarker discovery for interperson variability in sickle cell pain. This study exemplifies the potential of applying approaches from other pain conditions to advance the unmet needs of sickle cell pain. It also demonstrates the critical role of SCD pathobiology that evokes and maintains pain. The progressive inflammatory and nociceptive sickle cell microenvironment may continue to advance pain, thereby challenging the efficacy of analgesics that target the nervous system. Therefore, simultaneously targeting the SCD pathobiology and nociceptive mechanisms may improve the outcomes of analgesic therapies.

Conflict-of-interest disclosure: K.G. reports receiving research grants from Novartis, Grifols, Zilker LLC, and University of California, Irvine Foundation not related to this work. V.K. declares no competing financial interests.