Dynamic changes to lipid mediators support transitions among macrophage subtypes during muscle regeneration

Abstract

Muscle damage elicits a sterile immune response that facilitates complete regeneration. Here, we used mass spectrometry-based lipidomics to map the mediator lipidome during the transition from inflammation to resolution and regeneration in skeletal muscle injury. We observed temporal regulation of glycerophospholipids and production of pro-inflammatory lipid mediators (for example, leukotrienes and prostaglandins) and specialized pro-resolving lipid mediators (for example, resolvins and lipoxins) that were modulated by ibuprofen. These time-dependent profiles were recapitulated in sorted neutrophils and Ly6C^hi and Ly6C^lo muscle-infiltrating macrophages, with a distinct pro-resolving signature observed in Ly6C^lo macrophages. RNA sequencing of macrophages stimulated with resolvin D2 showed similarities to transcriptional changes found during the temporal transition from Ly6C^hi macrophage to Ly6C^lo macrophage. In vivo, resolvin D2 increased Ly6C^lo macrophages and functional improvement of the regenerating muscle. These results reveal dynamic lipid mediator signatures of innate immune cells and provide a proof of concept for their exploitable effector roles in muscle regeneration.

Figure 1.. Temporal regulation of structural and signaling lipids during tissue regeneration after sterile muscle injury.

(a) Representative images of H&E-stained TA muscles at days 0 and 8 after CTX-injury (at least 5 experiments were repeated independently with similar results). Scale bars are 100 μm. (b) Fiber size repartition of uninjured and regenerating muscle at day 8 post CTX injury (left panel). Data indicated mean ± SEM and p<0.05=*, p<0.001=***, p<0.0001=**** by Sidak’s multiple comparison test in two-way ANOVA. Mean (± SEM) fiber CSA is shown on the right panel. p<0.0001=**** by two-tailed unpaired Student’s t-test and n=at least 5 biologically independent muscle samples for both panels. (c) Relative distribution of lipid classes at selected time points after CTX, expressed as percentages. Color coding, names and abbreviations are shown as inset. (d) Quantification of each lipid class at indicated timepoints after CTX-injury. p<0.05=*, p<0.01=**, p<0.001=***, p<0.0001=**** by Dunnett’s multiple comparison test in two-way ANOVA. Data are shown as mean ± SEM and n=3 biologically independent muscle samples for each time point. (e) Cumulative lipid composition of four distinct groups of phosphatidyl choline (PC) species at indicated timepoints post CTX-injury. PC species groups contain Arachidonic Acid (AA), Docosahexaenoic Acid (DHA), Eicosapentaenoic Acid (EPA) or other PUFA chains at the sn-2 position of PC. (f) Levels of AA, EPA, and DHA at indicated timepoints after CTX-injury. p<0.05=*, p<0.01=**, p<0.001=***, p<0.0001=**** by Dunnett’s multiple comparison test in one-way ANOVA. Data are shown as mean ± SEM and n=3 biologically independent muscle samples for each time point.

Figure 2.. PUFA-derived lipid mediator profiles of TA muscle are temporally regulated following CTX-muscle injury.

(a) Representative multiple reaction monitoring (MRM) chromatograms of bioactive lipid mediators derived from DHA (blue), AA (orange) and EPA (green). 3 experiments were repeated independently with similar results. (b) Representative MS/MS spectra of RvD2 (left panel) and LXB₄ (right panel) with their diagnostic ion assignments and structures shown as inset (3 experiments were repeated independently with similar results). (c) Stacked histogram showing the cumulative levels of each specific class of lipid mediators at the indicated day after CTX injury. Constituent members for each group are: Leukotrienes - LTB₄, Δ6-trans-LTB₄, Δ6-trans,12-epi-LTB₄; Prostaglandins - PGD₂, PGE₂, PGF_2α, TxB₂; Lipoxins - 15R-LXA₄, LXB₄, LXA₅, LXB₅; D-series resolvins - 17R-RvD1, RvD2, 17R-RvD3, RvD4, RvD5; E-series resolvins - RvE1, RvE2, RvE3. (d) Heatmap displaying the relative abundance of individual lipid mediators at the indicated day post CTX. Each column represents the average of n=3 biologically independent TA muscle samples for each indicated time point. (e) Levels of selected AA (orange)-, DHA (blue)-, and EPA (green)-derived lipid mediators displayed in their biosynthetic pathways. Data shows mean ± SEM and n=3 biologically independent muscle samples for each indicated time point. (f) Ratio of LTB₄ to total SPM (15R-LXA₄, LXB₄, LXA₅, LXB₅, 17R-RvD1, RvD2, 17R-RvD3, RvD4, RvD5, 10S,17S-diHDHA, MaR1, RvE1, RvE2, RvE3) at indicated days post-CTX. *p<0.05 (two-tailed unpaired Student’s t-test). Data show mean ± SEM and n=3 biologically independent muscle samples per time point.

Figure 3.. Changes in PUFA metabolomes are conserved across pharmacological and physiological models of skeletal muscle injury.

(a, b) Interaction network pathway analyses of the docosahexaenoic (DHA), arachidonic (AA) and eicospentaenoic (EPA) acid bioactive metabolomes in TA muscle after CTX (a) or eccentric exercise-induced injury (b). The networks depict both the relative changes of each lipid mediator at day 1 compared to day 0 (color of circle) and the absolute abundance of the mediators at day 1 (size of circle). Compounds that were not detected in the analysis are shown in black, while those that were not included in the analysis are shown in grey. n=3 biologically independent muscle samples per group per time point. (c) Quantification of selected lipid mediators from the AA, DHA and EPA bioactive metabolomes depicted in their biosynthetic pathways after EE. Data show mean ± SEM. and n=3 biologically independent animals per time point (d) Ratio of LTB₄ to total SPM (LXA₄, 15R-LXA₄, LXB₄, 15R-LXB₄, 17R-RvD1, RvD2, 17R-RvD3, RvD4, RvD5, RvD6, PD1, 17R-PD1, 10S,17S-diHDHA, MaR2, RvE3) at days 1 and 8 post eccentric exercise injury. *p<0.05 (two-tailed unpaired Student’s t-test). Data show mean ± SEM and n=3 biologically independent muscles per time point.

Figure 4.. Lipid mediator profiles and gene expression of lipid mediator receptors and enzymes are characteristic of specific immune cells during skeletal muscle regeneration.

(a) Immunofluorescence analysis of desmin (red), F4/80 (green), and nuclei (blue) in TA muscles of WT mice at the indicated day post-CTX. Scale bars are 100 μm for days 1–4 and 50 μm for day 0 and 8. Images represent 5 independent experiments with similar results. (b) Number of infiltrating neutrophils (CD45⁺ Ly6G^hi Ly6C^int F4/80⁻), inflammatory macrophages (CD45⁺ Ly6C^hi Ly6G^lo F4/80^lo), and repair macrophages (CD45⁺ Ly6C^lo Ly6G⁻ F4/80^hi) in regenerating muscle from mice at the indicated day post-CTX. Data show mean ± SEM and n=3 biologically independent experiments per group. (c) Partial least squares-discriminant analysis (PLS-DA) two-dimensional scores plot showing clustering of samples in distinct populations based on their global lipid mediator profile and specific immune cell subset at each day post-CTX (n=3 biologically independent samples per group). (d) Heatmap showing the relative abundance of individual lipid mediators of sorted cell (neutrophils, Ly6C^hi and Ly6C^lo macrophages) populations at the indicated day post-CTX. Each column represents the mean of at least 3 samples for each cell population per time point. (e) The ratio of LTB₄ to total SPM, as described previously, in each cell population at the indicated day post CTX. Data show mean ± SEM and n=at least 3 per population per time point. (f) Venn diagram presenting the number of identified lipid mediators from whole muscle tissue and from the sorted immune cell populations (g) Normalized mRNA levels; visualized as log₁₀(norm.expr.), of selected genes involved in lipid metabolism and signaling from Ly6C^hi and Ly6C^lo macrophages of days 2 and 4 post CTX and PMNs RNA-seq analysis (Ward’s clustering algorithm and Euclidean measure distance; n=3 independent samples for each cell population at the indicated time points).

Figure 5.. RvD2 induces a unique transcriptional signature in macrophages that has similarities to macrophages during the transition from inflammation to resolution and regeneration.

(a) Heat map showing the relative expression pattern of the top 120 differentially expressed genes in control and RvD2 treated (4 hours) naïve bone marrow-derived macrophages (BMDMs). Fold change values are visualized as log₁₀(FC). (b) Gene ontology (GO) analysis of the genes that are differentially expressed in control and RvD2 treated BMDMs. Fold enrichment threshold was set at >2 and Bonferroni-corrected for p value < 0.05. (c) Venn Diagram showing the differentially expressed genes from control versus RvD2 treated BMDMs, integrated with the common differentially expressed (DE) genes in Ly6C macrophage subsets (see inset). In total, 172 genes are common between the two datasets, while 579 genes were DE only in RvD2 treated BMDMs. (d) Heatmaps showing normalized expression values of genes in Ly6C^hi and Ly6C^lo macrophages of days 2 and 4 post CTX that are common and DE between the two datasets (i.e., control vs RvD2 and Ly6C^hi vs Ly6C^lo at days 2/4). Hierarchical clustering analysis was performed for the transcript levels of the differentially expressed genes (Ward’s clustering algorithm and Euclidean measure distance and n=3 biologically independent samples for each cell populations at indicated time points).

Figure 6.. RvD2 administration increases the proportion of Ly6C^lo macrophages and improves in vivo force generation in a model of delayed muscle regeneration.

(a) Absolute number of infiltrating CD45⁺ cells in TA muscle of chimeric mice administered with saline (control) or RvD2 intramuscularly on day 2 or day 3 post CTX injury. Data show mean ± SEM and n=4 biologically independent samples per treatment group. (b) Representative flow cytometry contour plots of Ly6C^hi and Ly6C^lo macrophages from saline (control) and RvD2 treated animals at day 4 post CTX injury. Images represent 4 independent experiments with similar results. (c) Frequency of inflammatory (Ly6C^hi F4/80^lo) and repair (Ly6C^lo F4/80^hi) macrophages from saline (control) and RvD2 treated animals at day 4 post CTX-injury. p<0.05=*, p<0.01=**, p<0.001=***, p<0.0001=**** by Sidak’s multiple comparisons test in two-way ANOVA. Data are shown as mean ± SEM and n= at least 4 mice per group. (d). 2-DG uptake FLI signals, expressed as average radiance efficiency ([p·s⁻¹·cm⁻²·sr⁻¹]/[μW·cm⁻²]). Data are shown as mean ± SEM (n = 4 mice per group). (e) and (f) Quantification of in vivo muscle twitch (e) and tetanus forces (f). Data are shown as mean ± SEM (n= 4 mice per group) in saline and RvD2 treated chimeric mice at day 8 and day 14 post CTX. p<0.05=*, p<0.01=**, by Sidak’s multiple comparison test in two-way ANOVA.