IL-5 mediates monocyte phenotype and pain outcomes in fibromyalgia

Abstract

Fibromyalgia (FM) is characterized by widespread chronic pain, fatigue, and somatic symptoms. The influence of phenotypic changes in monocytes on symptoms associated with FM is not fully understood. The primary aim of this study was to take a comprehensive whole-body to molecular approach in characterizing relationships between monocyte phenotype and FM symptoms in relevant clinical populations. Lipopolysaccharide-evoked and spontaneous secretion of IL-5 and other select cytokines from circulating monocytes was higher in women with FM compared to women without pain. In addition, greater secretion of IL-5 was significantly associated with pain and other clinically relevant psychological and somatic symptoms of FM. Furthermore, higher levels of pain and pain-related symptoms were associated with a lower percentage of intermediate monocytes (CD14++/CD16+) and a greater percentage of nonclassical monocytes (CD14+/CD16++) in women with FM. Based on findings from individuals with FM, we examined the role of IL-5, an atypical cytokine secreted from monocytes, in an animal model of widespread muscle pain. Results from the animal model show that IL-5 produces analgesia and polarizes monocytes toward an anti-inflammatory phenotype (CD206+). Taken together, our data suggest that monocyte phenotype and their cytokine profiles are associated with pain-related symptoms in individuals with FM. Furthermore, our data show that IL-5 has a potential role in analgesia in an animal model of FM. Thus, targeting anti-inflammatory cytokines such as IL-5 secreted by circulating leukocytes could serve as a promising intervention to control pain and other somatic symptoms associated with FM.

Trial registration: ClinicalTrials.gov NCT01888640.

Copyright © 2020 International Association for the Study of Pain.

Figures

Figure 1.. LPS-stimulated human peripheral monocytes from participants with fibromyalgia (FM) and the No Pain (NP) group.

Graphs illustrate cytokine synthesis and secretion by isolated peripheral monocytes from participants with FM (red bars, n = 19) and NP participants (blue bars, n = 33). Cytokine Human Magnetic 10-Plex cytokine assay showed increased LPS-evoked synthesis and secretion of IL-5 (A), IL-2 (E), and -IL-4 (F) in FM participants compared with NP participants. There was less LPS-evoked synthesis and secretion of GM-CSF (D) and TNF-α (G). Data are presented as mean ± SEM. The Wilcoxon Rank-Sum Test with corrections for multiple comparisons was used to compare respective groups. * p

Figure 2.. Difference in monocyte subpopulations in between fibromyalgia (FM) and No Pain (NP) participants.

A) Gating strategy to isolate the monocyte population. B) Bar graphs indicate monocyte subpopulations (Classical, Intermediate and Non-Classical from (FM) participants (red bars, n = 32) and NP (blue bars, n = 37). Flow cytometry data analysis shows that women with FM had a higher percentage of Intermediate monocytes (CD14++/CD16+) and lower percentage of Non-Classical monocytes (CD14+/CD16++) compared with women without pain (NP). Data are represented as mean ± SEM. The Wilcoxon Rank-Sum Test with corrections for multiple comparisons was used to compare respective groups. * p<0.05 versus NP group.

Figure 3.. Assessment of pain like behavior and IL-5 expression in established experimental model of widespread muscle pain.

A) Represents the experimental timeline for gastrocnemius injection of normal and acidic saline on Day 0 (D0) and Day 5 (D5) to induce pain in the animal model of widespread muscle pain, and time point for the Von Frey testing to assess pain like behavior and flow cytometry analysis on Day 10 (D10) for IL5 expression. B) Line graph represents gastrocnemius injection of acidic saline on D0 and D5 on ipsilateral side induces the bilateral persistent pain line behavior (mechanical hypersensitivity) C. Flow cytometry analysis of peripheral monocytes isolated from the widespread muscle pain and control mice on D10. Gating strategy was applied to examine expression of IL-5 in different subpopulation of monocytes. Data are represented as mean ± SEM. n = 8 mice per group for hypersensitivity test (Von Frey testing) and n = 6 mice per group for flow cytometry experiment. Two-way AVOVA with Sidak’s multiple comparisons test was applied to compare two different groups. ***p

Figure 4.. Effect of IL-5 on widespread muscle pain behavior and monocyte polarization.

A) Maximal anti-allodynia was measured using three different doses of IL-5 (0.1 pg/mouse, 0.1 ng/mouse, n=6 and 0.1 μg/mouse, n=6). B) Intravenous injection of IL-5 (0.1 μg/mouse) reverses widespread musculoskeletal induced pain behavior in ipsilateral and contralateral hind paw. C) Gating strategy to isolate the monocyte population. D) Flow cytometry data analysis shows the intravenous injection of IL-5 polarize the peripheral monocytes into more non-classical (M2). Data are represented as mean ± SEM. n= 5-8 mice per group for IL-5 pharmacological study in normal and acidic saline injected mice. n=4-7 mice per group in normal and acidic saline injected mice after treatment of IL-5 for polarization-flow cytometry study. Two-way ANOVA with Dunnett’s post-test was applied to assess the anti-nociceptive effect of IL-5. Ordinary one-way ANOVA with Sidak’s multiple comparison test was applied to assess the flow cytometry data. *p