Longitudinal multisite study of the chronobiological control of chronic pain: the CircaHealth CircaPain study protocol.

INTRODUCTION: One in five Canadians lives with chronic pain. Evidence shows that some individuals experience pain that fluctuates in intensity following a circadian (24-hour) rhythm. Endogenous molecular rhythms regulate the function of physiological processes that govern pain mechanisms. Addressing chronic pain rhythmicity on a molecular and biopsychosocial level can advance understanding of the disease and identify new treatment/management strategies. Our CircaHealth CircaPain study uses an online survey combined with ecological momentary assessments and biosample collection to investigate the circadian control of chronic pain and identify potential biomarkers. Our primary objective is to understand interindividual variability in pain rhythmicity, by collecting biopsychosocial measures. The secondary objective accounts for seasonal variability and the effect of latitude on rhythmicity. METHODS AND ANALYSIS: Following completion of a baseline questionnaire, participants complete a series of electronic symptom-tracking diaries to rate their pain intensity, negative affect, fatigue and stress on a 0-10 scale at 8:00, 14:00 and 20:00 daily over 10 days. These measures are repeated at 6 and 12 months postenrolment to account for potential seasonal changes. We aim to recruit ≥2500 adults with chronic pain within Canada. Infrastructure is being developed to facilitate the collection of blood samples from subgroups of participants (~800) two times per day over 24-48 hours to identify rhythmic expression of circulating genes and/or proteins. ETHICS AND DISSEMINATION: Ethical approval for this study was obtained by the Queen's University Health Sciences and Affiliated Teaching Hospitals Research Ethics Board (File No. 6038114). Participants provide informed consent to participate, and their data will not be identifiable in any publication or report. Findings will be published in a relevant scientific journal and disseminated at scientific meetings and online webinars. We maintain a website to post updated resources and engage with the community. We employ knowledge mobilisation in the form of direct data sharing with participants.

Genome-wide association studies with experimental validation identify a protective role for B lymphocytes against chronic post-surgical pain.

BACKGROUND: Chronic post-surgical pain (CPSP) significantly impacts patients' recovery and quality of life. Although environmental risk factors are well-established, genetic risk remains less understood. METHODS: A meta-analysis of genome-wide association studies followed by partitioned heritability was performed on 1350 individuals across five surgery types: hysterectomy, mastectomy, abdominal, hernia, and knee. In subsequent animal studies, withdrawal thresholds to evoked mechanical stimulation were measured in Rag1 null mutant and wild-type mice after plantar incision and laparotomy. Cell sorting by flow cytometry tracked recruitment of immune cell types. RESULTS: We discovered 77 genome-wide significant single-nucleotide polymorphism (SNP) hits, distributed among 24 loci and 244 genes. Meta-analysis of all cohorts estimated a SNP-based narrow-sense heritability for CPSP at ∼39%, indicating a substantial genetic contribution. Partitioned heritability analysis across a wide variety of tissues revealed enrichment of heritability in immune system-related genes, particularly those associated with B and T cells. Rag1 null mutant mice lacking both T and B cells exhibited exacerbated and prolonged allodynia up to 42 days after surgery, which was rescued by B-cell transfer. Recruitment patterns of B cells but not T cells differed significantly during the first 7 days after injury in the footpad, lymph nodes, and dorsal root ganglia. CONCLUSIONS: These findings suggest a key protective role for the adaptive immune system in the development of chronic post-surgical pain.

The ion channel TRPV5 regulates B-cell signaling and activation.

Introduction: B-cell activation triggers the release of endoplasmic reticulum calcium stores through the store-operated calcium entry (SOCE) pathway resulting in calcium influx by calcium release-activated calcium (CRAC) channels on the plasma membrane. B-cell-specific murine knockouts of SOCE do not impact humoral immunity suggesting that alternative channels may be important. Methods: We identified a member of the calcium-permeable transient receptor potential (TRP) ion channel family, TRPV5, as a candidate channel expressed in B cells by a quantitative polymerase chain reaction (qPCR) screen. To further investigate the role of TRPV5 in B-cell responses, we generated a murine TRPV5 knockout (KO) by CRISPR-Cas9. Results: We found TRPV5 polarized to B-cell receptor (BCR) clusters upon stimulation in a PI3K-RhoA-dependent manner. TRPV5 KO mice have normal B-cell development and mature B-cell numbers. Surprisingly, calcium influx upon BCR stimulation in primary TRPV5 KO B cells was not impaired; however, differential expression of other calcium-regulating proteins, such as ORAI1, may contribute to a compensatory mechanism for calcium signaling in these cells. We demonstrate that TRPV5 KO B cells have impaired spreading and contraction in response to membrane-bound antigen. Consistent with this, TRPV5 KO B cells have reduced BCR signaling measured through phospho-tyrosine residues. Lastly, we also found that TRPV5 is important for early T-dependent antigen specific responses post-immunization. Discussion: Thus, our findings identify a role for TRPV5 in BCR signaling and B-cell activation.

Interactions between skin-resident dendritic and Langerhans cells and pain-sensing neurons.

Various immune cells in the skin contribute to its function as a first line of defense against infection and disease, and the skin's dense innervation by pain-sensing sensory neurons protects the host against injury or damage signals. Dendritic cells (DCs) are a heterogeneous population of cells that link the innate immune response to the adaptive response by capturing, processing, and presenting antigens to promote T-cell differentiation and activation. DCs are abundant across peripheral tissues, including the skin, where they are found in the dermis and epidermis. Langerhans cells (LCs) are a DC subset located only in the epidermis; both populations of cells can migrate to lymph nodes to contribute to broad immune responses. Dermal DCs and LCs are found in close apposition with sensory nerve fibers in the skin and express neurotransmitter receptors, allowing them to communicate directly with the peripheral nervous system. Thus, neuroimmune signaling between DCs and/or LCs and sensory neurons can modulate physiologic and pathophysiologic pathways, including immune cell regulation, host defense, allergic response, homeostasis, and wound repair. Here, we summarize the latest discoveries on DC- and LC-neuron interaction with neurons while providing an overview of gaps and areas not previously explored. Understanding the interactions between these 2 defence systems may provide key insight into developing therapeutic targets for treating diseases such as psoriasis, neuropathic pain, and lupus.

Protease-Induced Excitation of Dorsal Root Ganglion Neurons in Response to Acute Perturbation of the Gut Microbiota Is Associated With Visceral and Somatic Hypersensitivity.

BACKGROUND & AIMS: Abdominal pain is a major symptom of diseases that are associated with microbial dysbiosis, including irritable bowel syndrome and inflammatory bowel disease. Germ-free mice are more prone to abdominal pain than conventionally housed mice, and reconstitution of the microbiota in germ-free mice reduces abdominal pain sensitivity. However, the mechanisms underlying microbial modulation of pain remain elusive. We hypothesized that disruption of the intestinal microbiota modulates the excitability of peripheral nociceptive neurons. METHODS: In vivo and in vitro assays of visceral sensation were performed on mice treated with the nonabsorbable antibiotic vancomycin (50 µg/mL in drinking water) for 7 days and water-treated control mice. Bacterial dysbiosis was verified by 16s rRNA analysis of stool microbial composition. RESULTS: Treatment of mice with vancomycin led to an increased sensitivity to colonic distension in vivo and in vitro and hyperexcitability of dorsal root ganglion (DRG) neurons in vitro, compared with controls. Interestingly, hyperexcitability of DRG neurons was not restricted to those that innervated the gut, suggesting a widespread effect of gut dysbiosis on peripheral pain circuits. Consistent with this, mice treated with vancomycin were more sensitive than control mice to thermal stimuli applied to hind paws. Incubation of DRG neurons from naive mice in serum from vancomycin-treated mice increased DRG neuron excitability, suggesting that microbial dysbiosis alters circulating mediators that influence nociception. The cysteine protease inhibitor E64 (30 nmol/L) and the protease-activated receptor 2 (PAR-2) antagonist GB-83 (10 µmol/L) each blocked the increase in DRG neuron excitability in response to serum from vancomycin-treated mice, as did the knockout of PAR-2 in NaV1.8-expressing neurons. Stool supernatant, but not colonic supernatant, from mice treated with vancomycin increased DRG neuron excitability via cysteine protease activation of PAR-2. CONCLUSIONS: Together, these data suggest that gut microbial dysbiosis alters pain sensitivity and identify cysteine proteases as a potential mediator of this effect.

Navigating the blurred path of mixed neuroimmune signaling.

Evolution has created complex mechanisms to sense environmental danger and protect tissues, with the nervous and immune systems playing pivotal roles. These systems work together, coordinating local and systemic reflexes to restore homeostasis in response to tissue injury and infection. By sharing receptors and ligands, they influence the pathogenesis of various diseases. Recently, a less-explored aspect of neuroimmune communication has emerged: the release of neuropeptides from immune cells and cytokines/chemokines from sensory neurons. This article reviews evidence of this unique neuroimmune interplay and its impact on the development of allergy, inflammation, itch, and pain. We highlight the effects of this neuroimmune signaling on vital processes such as host defense, tissue repair, and inflammation resolution, providing avenues for exploration of the underlying mechanisms and therapeutic potential of this signaling.