Designed CXCR4 mimic acts as a soluble chemokine receptor that blocks atherogenic inflammation by agonist-specific targeting

Abstract

Targeting a specific chemokine/receptor axis in atherosclerosis remains challenging. Soluble receptor-based strategies are not established for chemokine receptors due to their discontinuous architecture. Macrophage migration-inhibitory factor (MIF) is an atypical chemokine that promotes atherosclerosis through CXC-motif chemokine receptor-4 (CXCR4). However, CXCR4/CXCL12 interactions also mediate atheroprotection. Here, we show that constrained 31-residue-peptides ('msR4Ms') designed to mimic the CXCR4-binding site to MIF, selectively bind MIF with nanomolar affinity and block MIF/CXCR4 without affecting CXCL12/CXCR4. We identify msR4M-L1, which blocks MIF- but not CXCL12-elicited CXCR4 vascular cell activities. Its potency compares well with established MIF inhibitors, whereas msR4M-L1 does not interfere with cardioprotective MIF/CD74 signaling. In vivo-administered msR4M-L1 enriches in atherosclerotic plaques, blocks arterial leukocyte adhesion, and inhibits atherosclerosis and inflammation in hyperlipidemic Apoe^-/- mice in vivo. Finally, msR4M-L1 binds to MIF in plaques from human carotid-endarterectomy specimens. Together, we establish an engineered GPCR-ectodomain-based mimicry principle that differentiates between disease-exacerbating and -protective pathways and chemokine-selectively interferes with atherosclerosis.

Fig. 1. The CXCR4 ectodomain mimic msR4M-L1 selectively binds to MIF but not CXCL12.

a Schematic summarizing the design strategy to utilize extracellular loop moieties of CXCR4 to engineer a soluble mimic that binds MIF but not CXCL12. b Ribbon structure of human CXCR4 based on the crystal structure according to PDB code 4RWS. Sequences of extracellular loops ECL1 and ECL2 that were found to interact with MIF according to peptide array mapping are highlighted in blue, and the N- and C-terminal residues of the ECL1 and 2 peptides are indicated. c Nanomolar affinity binding of msR4M-L1 to MIF as determined by fluorescence spectroscopic titrations. Emission spectra of Fluos-msR4M-L1 alone (blue; 5 nM) and with increasing concentrations of MIF at indicated ratios are shown (left panel; representative titration); binding curve derived from the fluorescence emission at 522 nm (right panel). d msR4M-L1 does not bind to CXCL12. Same as c but titration performed with increasing concentrations of CXCL12. e Conformation of CXCR4 ectodomain mimics as determined by far-UV CD spectroscopy. Mean residue ellipticity plotted over the wavelength between 195 and 250 nm. f–g Nanomolar binding of msR4M-L1 to MIF (f) but not CXCL12 (g) as determined by fluorescence polarization (FP). The FP signal of 5 nM Fluos-msR4M-L1 (as mP) is plotted over varied ligand concentration as indicated. h, i Binding of msR4M-L1 to MIF (h) but not CXCL12 (i) as confirmed by microscale thermophoresis (MST). The fraction of chemokine bound or normalized fluorescence change (ΔFnorm) to 100 nM TAMRA-msR4M-L1 is plotted against increasing concentrations of MIF or CXCL12, respectively. j Binding analysis between TAMRA-msR4M-L1 and MIF versus CXCL12 as determined by dot blot titration. Quantification from three independent blots (see representative blot in Supplementary Fig. 8). Data in c–d (right panel) and f–h are reported as means ± SD from three independent experiments; data in i are means ± SD from five independent experiments. Statistical analysis (j) was performed with two-way ANOVA and Sidak correction. CXCR4, CXC motif chemokine receptor-4; msR4M-L1, MIF-specific CXCR4 mimic-L1; MIF, macrophage migration-inhibitory factor. Source data are provided as a Source Data file.

Fig. 2. Mapping of the MIF/msR4M-L1 core binding region and complex disruption by mutations.

a Amino acid sequence of human MIF (boxed, top). The msR4M-L1 binding core region of MIF (sequence 38–80 and 54–80) is indicated in blue, while non-binding stretches are in gray (bottom). b Sequence of msR4M-L1. Aromatic residues identified by peptide array to be critical for MIF binding are highlighted in red. c–e Nanomolar affinity binding of msR4M-L1 to MIF(38–80) (c) and MIF(54–80) (d), but not MIF(6–23) (e), as determined by fluorescence spectroscopy. Emission spectra of Fluos-msR4M-L1 alone (blue; 5 nM) and with increasing concentrations of MIF(38–80) (c), MIF(54–80) (d), and MIF(6–23) (e) (left panels; representative titrations); binding curves derived from the fluorescence emission at 522 nm (right panels). f–g Binding of msR4M-L1 to MIF is blunted when aromatic residues in msR4M-L1 are substituted by Ala in analog msR4M-L1(7xAla) (f) or when N-loop residues in MIF are mutated to Ala in MIF(10xAla) (g). Fluorescence spectroscopy and binding curve as in c–e. Data in right panels of c–g are means ± SD from three independent experiments. h Dot blot shows that binding of TAMRA-msR4M-L1 to spotted MIF is attenuated by MIF(54–80). 400 ng spotted MIF was probed with TAMRA-msR4M-L1 +/− 2-fold molar excess of MIF(54–80); RFU, relative fluorescent units. The blot shown is one of three dot blots performed. msR4M-L1, MIF-specific CXCR4 mimic-L1; MIF, macrophage migration-inhibitory factor. Source data are provided as a Source Data file.

Fig. 3. msR4M-L1 selectively inhibits MIF-triggered CXCR4 activity, but spares the MIF/CD74 axis.

a, b MIF (a) but not CXCL12 (b) binding to and signaling through human CXCR4 in an S. cerevisiae system is attenuated by msR4M-L1 in a concentration-dependent manner. The molar excess of competing msR4M-L1 over MIF or CXCL12 is indicated. CXCR4 binding/signaling is read out by LacZ reporter-driven luminescence. c A 5-fold molar excess of msR4M-L1 does not interfere with binding of Alexa 488-MIF to CD74 expressed on HEK293-CD74 transfectants as measured by flow cytometry. Left, shift of CD74 transfectants following Alexa 488-MIF binding (control indicates background); right, quantification of three independent experiments. d, e Chemotactic migration (Transwell) of primary mouse spleen B lymphocytes elicited by 16 nM MIF (d) or CXCL12 (e) as chemoattractant and inhibitory effect of msR4M-L1. msR4M-L1 dose-dependently inhibits MIF-mediated chemotaxis (d), but the optimal inhibitory dose of 80 nM does not affect CXCL12-elicited chemotaxis (e). f msR4M-L1 analog msR4M-L1(7xAla) does not inhibit MIF-mediated chemotaxis. msR4M-L1(7xAla) was applied at a concentration of 80 nM. g msR4M-L1 does not interfere with MIF-triggered AMPK signaling in the human cardiomyocyte cell line HCM. MIF was applied at a concentration of 16 nM; msR4M-L1 added at 1- and 5-fold excess over MIF. AMPK signaling was measured using Western blot of HCM lysates developed against pAMPK and total AMPK. The densitometric ratio of pAMPK/AMPK indicates signaling intensity. Data are reported as means ± SD of n = 3 (a); n = 4 (b); n = 3 (c, right panel); n = 6 (d); n = 3 (e–f); and n = 5 (g) independent biological experiments. Statistical analysis was performed with unpaired two-tailed T-test. CXCR4, CXC motif chemokine receptor-4; msR4M-L1, MIF-specific CXCR4 mimic-L1; MIF, macrophage migration-inhibitory factor. Source data are provided as a Source Data file.

Fig. 4. msR4M-L1 specifically inhibits MIF- but not CXCL12-elicited atherogenic monocyte activities.

a, b MIF-mediated DiI-oxLDL uptake in primary human monocyte-derived macrophages is dose-dependently inhibited by msR4M-L1 (indicated as molar excess over MIF). MIF was applied at a concentration of 80 nM. a Representative images of DiI-oxLDL-positive cells; b quantification (three-times-two independent experiments; 9 fields-of-view each). c, d MIF-specific DiI-LDL uptake in primary human monocyte-derived macrophages is dose-dependently inhibited by msR4M-L1 (indicated as molar excess over MIF) (c), but not by the MIF binding-dead analog of msR4M-L1, msR4M-L1(7xAla) (d). MIF was applied at a concentration of 80 nM. Quantification (four-times-two or three-times-two plus one-time-three, respectively, independent experiments; 9 fields-of-view each). AMD3100 (AMD) was used to verify CXCR4 dependence of the MIF effect. e Same as in c, d, except that the small molecule inhibitor ISO-1 and neutralizing MIF antibody NIH/IIID.9 were used instead of msR4M-L1 (three-times-two independent experiments; 9 fields-of-view each; isotype control antibody IgG1: two-times-two). f, g Representative experiment demonstrating that msR4M-L1 inhibits MIF-elicited (red tracks) 3D chemotaxis of human monocytes as assessed by live-microscopic imaging of single-cell migration tracks in x/y direction in µm. Increasing concentrations of msR4M-L1 (blue tracks, molar excess over MIF) as indicated; unstimulated control (gray tracks) indicates random motility. i Quantification of f, g; the migration tracks of 32–37 randomly selected cells per treatment group were recorded and the forward migration index plotted; the experiment shown is one of three independent experiments with monocytes from different donors. h A 5-fold molar excess of msR4M-L1 does not affect 3D human monocyte migration elicited by CXCL12; j quantification of h; the migration tracks of 29–30 randomly selected cells per treatment group were recorded and the forward migration index plotted; the experiment shown is one of two independent experiments with monocytes from different donors. Data in b–e, i, and j are reported as means ± SD. Statistical analysis was performed with one-way ANOVA with Tukey’s multiple comparisons test or Kruskal–Wallis with Dunn’s multiple comparisons test. The scale bar in a is: 50 µm. CXCR4, CXC motif chemokine receptor-4; msR4M-L1, MIF-specific CXCR4 mimic-L1; MIF, macrophage migration-inhibitory factor. Source data are provided as a Source Data file.

Fig. 5. msR4M-L1 localizes to atherosclerotic plaques in a MIF-specific way and inhibits atherogenic leukocyte arrest.

a, b MIF-induced static adhesion of MonoMac-6 monocytes to HAoECs is ablated by msR4M-L1. a Resting HAoECs. b As in a, except that HAoECs were pre-incubated with TNF-α. Quantification based on 3 experiments with 10 independent fields-of-view each. c MIF-induced adhesion of MonoMac-6 monocytes to HAoECs under flow conditions (1.5 dyn/cm²) is ablated by msR4M-L1. One of two independent experiments with four analyses each. d, e Fluos-msR4M-L1 stains aortic root sections from atherogenic Ldlr^−/− mice on HFD in a MIF-specific manner (comparison between Ldlr^−/− and Ldlr^−/−Mif^−/− mice). d Representative images (PC, phase contrast; DAPI, nuclei); e quantification (relative fluorescence units) from two independent experiments with two animals each indicates MIF-specific staining. f, g Multiphoton laser-scanning microscopy image (f) of a carotid artery prepared from a hyperlipidemic Apoe^−/− mouse, showing that in vivo administered Fluos-msR4M-L1 localizes to atherosclerotic plaques. Vessel visualization by second harmonic generation (SHG). g Quantification of the Fluos-msR4M-L1-positive area (means of n = 3 sections) as percentage of vessel target-area. h (and g) Same as f, g, except that aortic root was prepared (green in upper panel is autofluorescence). Quantification in g indicates ORO-positive target-area. i–l msR4M-L1 inhibits leukocyte adhesion in atherogenic carotid arteries under flow as analyzed by MPM. i Schematic summarizing the ex vivo leukocyte adhesion experiment. msR4M-L1 or vehicle was injected before vessel harvest; flushed leukocytes are stained in red (msR4M-L1; CMPTX) or green (vehicle; CMFDA). j Representative image of a carotid artery showing that pre-treatment with msR4M-L1 (red) leads to reduced luminal leukocyte adhesion compared to control (green), imaged by 3D reconstruction after Z-stacking (0.8–1.5 µm) (blue: SHG). k Still image of a z sectioning video scan (single field of view) through the artery (morphology revealed by SHG: collagen, dark blue; elastin, light blue). l Quantification of 5–6 independent carotid arteries per group. Luminally-adhering cell numbers are plotted. Scale bars: d, 50 µm; f, 100 µm; h, 100 µm; j–k, 100 µm. Data in a, b, c, e, g and l are reported as means ± SD. Statistical analysis was performed with one-way ANOVA with Tukey’s multiple comparisons test or two-tailed Mann–Whitney test as appropriate. The vessel icon in Fig. 5i was used with permission from S. Karger AG (Copyright © 2006, © 2007 S. Karger AG, Basel). msR4M-L1, MIF-specific CXCR4 mimic-L1; MIF, macrophage migration-inhibitory factor. Source data are provided as a Source Data file.

Fig. 6. msR4M-L1 inhibits atherosclerosis in vivo and msR4M-L1-based staining parallels MIF expression in human plaques.

a SDS-PAGE imaging of TAMRA-msR4M-L1 mouse plasma incubations verifies proteolytic stability (red bands, TAMRA-msR4M-L1; green, running-dye). b In vivo stability of msR4M-L1. TAMRA-msR4M-L1 i.p.-injected into C57BL/6 mice and plasma collected as indicated. The pharmacokinetic is derived from SDS-PAGE TAMRA-msR4M-L1 bands (inset) applying a TAMRA-msR4M-L1 calibration curve (Supplementary Fig. 23). c Schematic showing the in vivo injection regimen in atherogenic Apoe^−/− mice. d, e msR4M-L1 treatment reduces atherosclerotic lesions in aortic arch. Representative images (d) and quantification (e) of HE-stained sections from 7 msR4M-L1- versus 7 vehicle-treated mice. f, g msR4M-L1 treatment reduces lesions in aortic root. Representative images (f) and quantification (g) of ORO-stained sections from 12 msR4M-L1- versus 12 vehicle-treated mice. h, i msR4M-L1 treatment reduces macrophage content in aortic root. Representative images (h) and quantification of macrophage area (i) of anti-MAC2-stained (red) sections from 11 msR4M-L1- versus 12 vehicle-treated mice. j msR4M-L1 reduces circulating inflammatory cytokines. Cytokine-array analysis of plasma samples from 6 msR4M-L1- versus 6 vehicle-treated Apoe^−/− mice on HFD (duplicates each) (means ± SD). k–m MIF gene expression is upregulated in atherosclerotic plaques from CEA patients compared to healthy vessels but no difference between plaque stages. Expression measured with mRNA from paraffin sections (k, qPCR, n = 19 stable and 20 unstable plaques, n = 4 healthy vessels) and mRNA from fresh tissue (l, qPCR, n = 9 early, n = 9 advanced plaques; m, RNAseq, n = 6 stable and n = 5 unstable plaques). n–p Side-by-side-comparison (representative images) between anti-MIF antibody-based IHC and TAMRA-msR4M-L1 staining for selected sections of stable CEA plaques (n, n = 6 sections, 11 specimens), unstable plaques (o, n = 2 sections, 15 specimens), and healthy vessel (p, n = 2 sections, 6 specimens); (left, overviews; right, magnifications of selected areas (boxes 1, 2) stained by anti-MIF antibody (brown) and TAMRA-msR4M-L1 probe (red), DAPI + (blue); circular arrow indicates DAB-/TAMRA-stained slides are not in same orientation). Quantification of TAMRA-msR4M-L1-stained full cohorts in Supplementary Fig. 27b. Scale bars: d 50 µm; f 200 µm; h 200 µm; n–p 100 µm. Data in e, g, i, and j are reported as means ± SD; data in k–m as box-whisker plots. Statistical analysis performed with unpaired two-tailed t-test (e, g, i, j), two-tailed Mann–Whitney test, or Kruskal–Willis test (k, l, m) as appropriate. msR4M-L1, MIF-specific CXCR4 mimic-L1; MIF, macrophage migration-inhibitory factor. Source data including definitions of box-plot parameters (minima, percentiles, centers, maxima) of k–m are provided as a Source Data file.