Extended Data Fig. 7: NAMPT binds to the CCR5 receptor present on MuSCs and induces proliferation.

a, b, NAMPT selectively binds to CCR5. a, ELISA plates were coated with human recombinant CCR5 (hrCCR5) or BSA and further incubated with hrNAMPT₍₁₎ at increasing concentrations (0 nM to 800 nM). NAMPT molecules bound to CCR5 were detected using a biotinylated antibody. NAMPT binds to CCR5 with a K_D of 172 ± 18 nM (n = 2 independent experiments performed in triplicate). The graph shows a representative binding curve from which non-specific binding to BSA was deducted. Data are mean ± s.e.m. b, NAMPT is a highly evolutionary conserved protein, with human NAMPT sharing 96% and 88.24% identity with mouse NAMPT and zebrafish Nampta proteins, respectively. As we use hrNAMPT in mouse cell line assays, we also assayed the affinity of hrNAMPT for mrCCR5 by means of a competitive ligand-binding assay with the cognate ligand, CCL4. ELISA plates were coated with mrCCR5 or BSA and incubated with mrCCL4 at increasing concentrations (0 nM to 400 nM) along with 100 nM hrNAMPT₍₁₎. hrNAMPT₍₁₎ molecules bound to CCR5 were detected using a biotinylated antibody. mrCCL4 shows an IC₅₀ of 34.4 ± 2.2 nM (n = 2 independent experiments performed in triplicate) revealing both hrNAMPT’s strong affinity for mrCCR5 and its binding to the receptor via the same sites used by CCL4. The graph shows a combined binding curve from which non-specific binding to BSA was deducted. Data are mean ± s.e.m. c, Exogenous NAMPT supplementation enhances myoblast proliferation. In vitro assay assessing the effects of exogenously introduced factors on C2C12 myoblast proliferation. Proliferation is identified by EdU incorporation. NAMPT administration (two commercially available NAMPT sources tested, hrNAMPT₍₁₎ and hrNAMPT₍₂₎) leads to a dose-dependent increase in myoblast proliferation. This effect is specifically mediated by the CCR5 receptor. Co-administration of NAMPT with the CCR2/CCR5 dual inhibitor CVC and CCR5-specific inhibitor MVC abolishes the pro-proliferative response of NAMPT, whereas co-administration with the CCR2 inhibitor PF-4136309 (PF4) does not hinder the stimulatory effect of NAMPT on myoblast proliferation. In agreement with this finding, the endogenous ligands of CCR5, mrCCL8 and mrCCL4, functioned to enhance C2C12 proliferation, whereas the CCR2-specific ligand mouse recombinant CCL2 did not to increase proliferative rates beyond that of the control. The pro-proliferative function of NAMPT is separate from its intracellular role in energy metabolism, as co-administering NAMPT with the NAMPT enzymatic inhibitor (GMX1778) did not affect its effect on myoblast proliferation. The central lines, upper and lower lines within the violin plot indicate the median, upper and lower quartiles, respectively. Two-way ANOVA with Tukey’s multiple comparison test. n = 20 control, n = 10 1.9 nM hrNAMPT₍₁₎, n = 10 9.5 nM hrNAMPT₍₁₎, n = 10 19 nM hrNAMPT₍₁₎, n = 9 1.9 nM hrNAMPT₍₂₎, n = 10 9.5 nM hrNAMPT₍₂₎, n = 10 100 nM CVC, n = 10 100 nM MVC, n = 10 100 nM PF4, n = 10 100 nM CVC + 9.5 nM hrNAMPT₍₁₎, n = 10 100 nM MVC + 9.5 nM hrNAMPT₍₁₎, n = 9 100 nM PF4 + 9.5 nM hrNAMPT₍₁₎, n = 10 9.5 nM mrCCL8, n = 10 9.5 nM mrCCL4, n = 10 9.5 nM mrCCL2, n = 10 500 nM GMX1778, n = 10 500 nM GMX1778 + 9.5 nM hrNAMPT₍₁₎. d, Cultured MAF DKO macrophages actively secrete NAMPT. ELISA-quantified NAMPT concentration in the supernatants of 16-h M-CSF-stimulated MAF DKO and Raw 264.7 macrophages (n = 1 experiment in triplicate, stimulated with 10 ng ml⁻¹ M-CSF). The continuous lines and dotted lines within the violin plot indicate the median and quartiles, respectively. Unpaired two-tailed t-test; t₄ = 0.3302, P = 0.7578. e, f, Cultured MAF DKO macrophages do not actively secrete the cognate ligands of CCR5, CCL3, CCL4 and CCL5. e, Representative array spots detecting CCR5 ligands expressed by MAF DKO macrophages after 16 h stimulation with M-CSF (10 ng ml⁻¹). f, Quantification. Data are mean ± s.e.m. (n = 1 array per group performed in duplicate). g, h, PAX7⁺ satellite cells in mouse primary myoblast monocultures display enhanced proliferation upon CCR5 receptor signalling, mediated by either exogenous NAMPT (g, h) or CCL4 (h) supplementation. Coculturing myoblasts with macrophages stimulated satellite cell proliferation. This pro-proliferative response is abolished by administrating CVC (g) and MVC (h), whereas PF-4136309 had no negative effect (h), confirming that the pro-proliferative function of macrophages on stem cells is mediated by CCR5 signalling. In addition, coculturing myoblasts with 3T3 cells that do not naturally secrete NAMPT⁸³ does not stimulate satellite cell proliferation, suggesting that NAMPT is the macrophage-derived pro-proliferative cue that drives stem cell proliferation. g, n = 18 control, n = 18 9.5 nM hrNAMPT₍₂₎, n = 18 100 nM CVC, n = 18 100 nM CVC + 9.5 nM hrNAMPT₍₂₎ for myoblasts, myoblast + macrophages and myoblasts + 3T3 cells); h, n = 35 control, n = 36 9.5 nM hrNAMPT₍₁₎, n = 36 100 nM MVC, n = 36 100 nM PF4, n = 55 9.5 nM mrCCL4 for myoblasts, n = 42 control, n = 36 9.5 nM hrNAMPT₍₁₎, n = 36 100 nM MVC, n = 31 100 nM PF4, n = 46 9.5 nM mrCCL4 for myoblast + macrophages and n = 36 control, n = 36 9.5 nM hrNAMPT₍₁₎, n = 36 100 nM MVC, n = 36 100 nM PF4, n = 51 9.5 nM mrCCL4 for myoblast + 3T3 cells. The black lines and grey lines within the violin plot indicate the median and quartiles, respectively. Two-way ANOVA with Tukey’s multiple comparison test. i–m, Inhibiting Ccr5 receptor activation does not affect injury-responsive immune cell dynamics in larval zebrafish. i, j, The predicted orthologous Ccr5 protein in zebrafish was identified by determining the appropriate best hit for the protein most similar to the human CCR5 amino acid sequence in zebrafish using BLAST. A maximum-likelihood phylogenetic tree constructed using protein sequences of CCR5 positioned the putative zebrafish Ccr5-like (Ccr5l) sequence as a homologue of mammalian CCR5 with strong bootstrap support. Bootstrap values with 500 replicates are documented below the branches. CCR7 was used as an outgroup. The accession numbers for genes included in our analysis are provided in the table (j). k, pax3a⁺ MuSCs express the ccr5 receptor at 2 dpi as detected by RT–PCR (n = 3). l, m, Larvae soaked in CVC or MVC displayed a marked regeneration deficit revealed by birefringence imaging (l; scale bar, 500 μm) and quantification (m; n = 20 control, n = 22 5 μM CVC, n = 14 10 μM CVC, n = 16 5 μM MVC, n = 12 10 μM MVC). Individual data points are shown. Two-way ANOVA with Tukey’s multiple comparison test. n–s, Ccr5 inhibition by CVC does not affect macrophage (Tg(mpeg1:GAL4FF/UAS:nfsb-mCherry); yellow) migration into the injury site (skeletal muscle labelled using Tg(actc1b:BFP); magenta) or the successful transition into a dwelling macrophage subtype (n; scale bar, 50 μm). o, Quantification (n = 10 per group at each time point). The continuous lines and dotted lines within the violin plot indicate the median and quartiles, respectively. Two-way ANOVA with Tukey’s multiple comparison test. CVC-treated macrophages appear morphologically indistinguishable from controls, with transient macrophages possessing lower sphericity values then their dwelling counterpart (p; scale bar, 20 μm). q, Quantification (n = 45 transient control, n = 42 transient 5 μM CVC, n = 50 dwelling control, n = 39 dwelling 5 μM CVC). Data are mean ± s.d. Two-way ANOVA with Tukey’s multiple comparison test. r, CVC treatment does not alter the response of neutrophils (Tg(mpx:eGFP); magenta) to needle-stab muscle injury (white dotted line). Scale bar, 50 μm. s, Quantification (n = 11 control, n = 14 5 μM CVC). The continuous lines and dotted lines within the violin plot indicate the median and quartiles, respectively. Two-way ANOVA with Tukey’s multiple comparison test. Unpaired two-tailed t-test; t₂₃ = 0.5838, P = 0.5650. t–u, pax3a⁺ myogenic stem cell (TgBAC(pax3a:GFP); cyan) proliferation is inhibited by CVC addition as demonstrated by decreased EdU incorporation (magenta) of these cells in the wound site (white dotted line) after injury (t; scale bar, 50 μm) and quantification (u, n = 19 per group). The black lines and grey lines within the violin plot indicate the median and quartiles, respectively. Two-way ANOVA with Tukey’s multiple comparison test. Two-way ANOVA with Tukey’s multiple comparison test. v, CVC treatment (pre-treatment for 2 h and maintained after laser ablation skeletal muscle injury (white dotted line) until experimental end point) does not interfere with the initiation and maintenance of dwelling macrophage (white arrows)–MuSC (white arrowheads) associations in the wound site. Scale bar, 50 μm. Frames were obtained from Supplementary Video 13.

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