Fig 2: Localization of CXCL11 expression in developing and adult limb muscles. Sections were obtained from the quadriceps muscle of E18 a, b and adult rats c, d and double-labelled with antibodies against CXCL11 (red) and MyHC (green). Cell nuclei were visualized with DAPI. At each developmental stage, first row a, c and second row b, d show low and high magnification, respectively. a″–d″ Merging of immunocytochemical stainings and DAPI. In E18 muscles, CXCL11-immunolabelling was associated with many muscle fibres (arrows in a″). Higher magnification b″ confirmed the predominant intracellular localization of CXCL11 at this developmental stage. In adult muscles, CXCL11 expression was confined to cells within the endomysium/perimysium (arrow heads in d). Scale bars, a and c, 100 µm; b and d, 25 µm

Fig 3: CXCL11 does not affect anabolic and catabolic markers in C2C12 cells. a Undifferentiated C2C12 cells or a' cells differentiated for 3 days were treated with CXCL11 (100 ng/ml) for the indicated time and subsequently analysed for phosphorylated (activated) p70S6K by Western blotting using phospho-specific antibodies. To control for protein loading, blots were additionally stained with antibodies recognizing p70S6K independent of its phosphorylation status. CXCL11 does neither activate p70S6K in undifferentiated nor differentiated C2C12 cells. b C2C12 cells differentiated for 3 days with low serum DMEM were maintained for another 1–3 days with low serum DMEM in the absence or presence of CXCL11 (100 ng/ml) without further medium change (starvation condition) but daily renewal of CXCL11 and analysed for MuRF-1 expression by Western blotting. GAPDH served as a loading control. b' Results from densitometric analysis of immunoreactive protein bands (mean ± SD, n = 3–4) corrected for protein loading. MuRF-1 expression continuously increased in starving C2C12 cells/myotubes and remained unaffected by CXCL11. ap < 0.001, day 3 vs day 1; bp < 0.001, day 3 vs. day 2

Fig 4: Summary of pathological role of CXCR7 and potential benefit of CXCR7 antagonism during ALI/ARDS. (A) CXCR7 functions predominantly as a scavenger receptor for its two ligands: the interferon-inducible chemokine CXCL11 and the constitutive chemokine CXCL12. Binding of its ligands leads to internalization of the CXCR7–ligand complex and ligand degradation. CXCL11 and CXCL12 also bind and activate the signaling chemokine receptors CXCR3 and CXCR4, respectively. The CXCR3/CXCR4/CXCR7 axes play an important role in lung inflammation. CXCR7 scavenging activity tightly regulates the extracellular levels of its ligands, facilitating the establishment and maintenance of CXCL11/12 chemokine concentration gradients and CXCR3+/CXCR4+ cell migration from the blood to the inflamed lung. In addition, increased CXCR7 expression in the inflamed lung has been reported to be associated with breathing pattern alteration and endothelial barrier dysfunction. (B) CXCR7 antagonism with the CXCR7 antagonist ACT-1004-1239 exhibits immunomodulatory effects: by blocking the scavenging activity of the receptor and consequently increasing CXCL11 and CXCL12 plasma concentrations, chemokine gradients are disrupted, inhibiting CXCR3+ and CXCR4+ cell migration to the inflamed lung. In addition, treatment with ACT-1004-1239, by increasing plasma CXCL12 and/or by direct inhibition of CXCR7 signaling, ameliorates ALI-induced breathing pattern alteration and endothelial barrier dysfunction.

Fig 5: Kinetics of the expression of CXCR3/CXCR4 and its ligands in the BAL of LPS-challenged DBA/1 mice. ALI was induced by nebulized LPS inhalation in male DBA/1 mice. Control mice inhaled NaCl 0.9% (n = 12 mice; all time points were pooled). (A) LPS inhalation increased protein concentrations of the CXCR3 ligands CXCL9, CXCL10, and CXCL11, measured in the BAL 5, 24, 48, and 72 h following LPS challenge, compared with control mice. Results are expressed as mean ± SEM (n = 8 mice per time point). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 versus control mice using Student t-test. (B) Representative gating strategy for CXCR3+ myeloid and lymphoid cells. (C) CXCR3 expression on BAL lymphoid and myeloid-infiltrating cells. Results are expressed as mean ± SEM of the mean fluorescence intensity (MFI) of CXCR3 obtained for each LPS-challenged mouse (n = 7–8 mice per time point) corrected by the MFI obtained in the fluorescence minus one (FMO) controls for CXCR3. Negative MFI values were set to 0. The complete gating strategy for lymphoid and myeloid cells is illustrated in Supplementary Figure S3. (D) Proportion of CXCR3+ lymphoid and myeloid cells expressed as percentages (mean ± SEM) of the CD45+CD11b− and CD45+CD11b+ parent population, respectively, in the BAL (see gating strategy in Supplementary Figure S3) (n = 7–8 mice per time point). (E) Time course of BAL CXCR3+ lymphoid and myeloid cell infiltrates. Results are expressed as absolute counts in the BAL (mean ± SEM). **p < 0.01, ***p < 0.001, ****p < 0.0001 using Student t-test versus control mice. (F) LPS inhalation increased protein concentrations of the CXCR4 ligand CXCL12, measured in the BAL 5, 24, 48, and 72 h following LPS nebulization compared with control mice. Results are expressed as mean ± SEM (n = 7–8 mice per time point). **p < 0.01, ****p < 0.0001 versus control mice using Student t-test. (G) Representative gating strategy for CXCR4+ myeloid and lymphoid cells. (H) CXCR4 expression on BAL lymphoid and myeloid-infiltrating cells. Results are expressed as mean ± SEM of the MFI of CXCR4 obtained for each LPS-challenged mouse (n = 7–8 mice per time point) corrected by the MFI obtained in the FMO controls for CXCR4. Negative MFI values were set to 0. The gating strategy for lymphoid and myeloid cells is illustrated in Supplementary Figure S3. (I) Proportion of CXCR4+ lymphoid and myeloid cells expressed as percentages (mean ± SEM) of the CD45+CD11b− and CD45+CD11b+ parent population, respectively, in the BAL (see gating strategy in Supplementary Figure S3) (n = 7–8 mice per time point). (H) Time course of BAL CXCR4+ lymphoid and myeloid cell infiltrates. Results are expressed as absolute counts in the BAL (mean ± SEM) (n = 7–8 mice per time point). **p < 0.01, ***p < 0.001, ****p < 0.0001 using Student t-test versus control mice.