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P2X4 Receptors on Muscle Macrophages Are Required for Development of Hyperalgesia in an Animal Model of Activity-Induced Muscle Pain

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Abstract

Activity-induced pain is common in those with chronic musculoskeletal pain and limits participation in daily activities and exercise. Our laboratory developed a model of activity-induced pain and shows that depletion of muscle macrophages prevents development of hyperalgesia. Adenosine triphosphate (ATP) is released from fatiguing muscle and activates purinergic receptors (P2X), and P2X4 receptors are expressed on macrophages. We hypothesized that exercise releases ATP to activate P2X4 receptors on muscle macrophages, which subsequently release interleukin-1β (IL-1β) to produce hyperalgesia. In an animal model of activity-induced pain, using male and female C57BL6/J mice, we show increased expression of P2X4 on muscle macrophages, and blockade of P2X4 receptors in muscle prevented development of hyperalgesia. Using a lentivirus expressing an artificial micro-RNA to P2X4 under the control of a CD68 promoter, we decreased expression of P2X4 mRNA in cultured macrophages, decreased expression of P2X4 protein in muscle macrophages in vivo, and prevented development of activity-induced hyperalgesia. We further show that macrophages primed with LPS differentially released IL-1β when treated with ATP in neutral or acidic pH. Lastly, blockade of IL-1β in muscle prevented development of hyperalgesia in this model. Thus, our data suggest that P2X4 receptors could be a valid pharmacological target to control activity-induced muscle pain experienced by patients with chronic musculoskeletal pain.

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Acknowledgments

The authors would like to thank Dr. Steven P. Wilson, Professor Emeritus, University of South Carolina, for help with design, development, and writing of methods for using of the lentiviral vector.

Funding

This study was supported by the National Institute of Arthritis, Musculoskeletal and Skin Diseases (NIAMS) AR061371 and AR073187, the Carver College of Medicine at the University of Iowa, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001, and the Sao Paulo Research Foundation (FAPESP) [grant number 2014/01119-4].

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Authors and Affiliations

  1. School of Applied Sciences, State University of Campinas-UNICAMP, Limeira, 13484-350, Brazil

    Maria Claudia Oliveira-Fusaro

  2. Neuroscience Graduate Program, Pain Research Program, Department of Physical Therapy and Rehabilitation Science, The University of Iowa, Iowa City, IA, 52242, USA

    Nicholas S. Gregory, Sandra J. Kolker, Lynn Rasmussen & Kathleen A. Sluka

  3. Departments of Internal Medicine and Microbiology and Immunology, The University of Iowa, Iowa City, IA, 52242, USA

    Lee-Ann H. Allen

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  1. Maria Claudia Oliveira-Fusaro
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Contributions

Maria Claudia Oliveira-Fusaro designed the project, performed and analyzed behavioral experiments and some of immunofluorescence assays, collected tissue samples, wrote the manuscript; Nicholas S. Gregory provided support during fatigue experiments, collected tissue samples, designed lentivirus; Lynn Rasmussen provided support during behavioral experiments, performed and analyzed some of immunofluorescence assays; Sandra J. Kolker performed all experiments and analysis using cultured macrophages and images from some immunofluorescence assays; Lee-Ann H. Allen provided training and support for cytokine assays from cultured macrophages, design, and revised the manuscript, Kathleen A. Sluka conceived the study, was in charge of overall direction and planning, analyzed all final data, wrote and revised the manuscript. All authors provided critical feedback and contributed to the final manuscript.

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Correspondence to Kathleen A. Sluka.

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Supplementary Fig1

Behavioral data analyzed for sex differences. a,b. Scatter plots with mean ± S.E.M. showing withdrawal thresholds of the ipsilateral (a) and contralateral (b) muscle 24h after induction of the model for male (blue) and female (red) mice treated with vehicle or 5, 50, or 500μM 5-BDBD on Day 5 prior to the fatiguing contraction and second intramuscular saline injection. For comparison withdrawal threshold from the vehicle treated mouse prior to induction of the model are shown (gray, N). +, p<0.05 compared to vehicle. c.d. Scatter plots mean ± S.E.M. showing withdrawal thresholds of the ipsilateral (c) and contralateral (d) muscle 24h after induction of the model for male (blue) and female (red) mice treated with vehicle or 500μM 5-BDBD prior to the 1st intramuscular saline injection. For comparison withdrawal threshold from the vehicle treated mouse prior to induction of the model are shown (gray, N). +, p<0.05 compared to vehicle. e,f. Scatter plots mean+S.E.M. showing withdrawal thresholds of the ipsilateral (e) and contralateral (f) muscle 24h after induction of the model for male (blue) and female (red) mice treated with a lentivirus expressing a control miRNA (miR_C) or an artificial miRNA to P2X4 (miR_P2X4) prior to the 1st intramuscular saline injection. For comparison withdrawal threshold from the vehicle treated mouse prior to induction of the model are shown (gray, N). +, p<0.05 compared to vehicle. Data are represented as the mean and SEM. (JPG 58 kb)

Supplementary Fig2

Effects of ATP, pH and LPS on release of cytokines from cultured macrophages. Scatter plots with mean and SEM for GM-CSF (a), IL-5 (b), IL-4 (c), IFNγ (d), IL-10 (e), and TNFα (f) released from cultured peritoneal macrophages treated different combinations of LPS, ATP or acidic pH. ATP evoked release of GM-CSF with or without acidic pH, LPS evoked release of IL-5 and IL-4 regardless of treatment with ATP or pH, and there were no consistent changes in IFNγ, IL-10 or TNFα. * P<0.05 compared to LPS-/ATP-/pH7.4 condition. (JPG 90 kb)

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Oliveira-Fusaro, M.C., Gregory, N.S., Kolker, S.J. et al. P2X4 Receptors on Muscle Macrophages Are Required for Development of Hyperalgesia in an Animal Model of Activity-Induced Muscle Pain. Mol Neurobiol 57, 1917–1929 (2020). https://doi.org/10.1007/s12035-019-01852-x

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