Fig 2: 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 3: Antagonism of CXCR7 increases plasma CXCL11 and CXCL12 levels and decreases CXCR3+ and CXCR4+ BAL infiltrates post LPS challenge. ALI was induced by nebulized LPS inhalation, and DBA/1 mice were treated with vehicle (LPS-vehicle, black bars) or ACT-1004-1239 (LPS-ACT-1004-1239, 100 mg/kg, red bars) orally, twice daily, 1 h prior to LPS challenge. Control mice received vehicle 1 h prior to NaCl 0.9% inhalation (NaCl-vehicle, white bars; n = 12 mice, pool of all time points). Protein concentrations of CXCL11 and CXCL12 in the plasma and lung tissue and BAL flow cytometry of immune infiltrates were performed 24, 48, and 72 h after challenge (n = 6–8 mice per time point). Time course of CXCL11 protein concentration in the plasma (A) and lung tissue (B). Chemokine concentrations are expressed in pg/ml (plasma) or pg/lung homogenate (mean ± SEM). **p < 0.01, ***p < 0.001 versus LPS-vehicle-treated animals or # p < 0.05, ### p < 0.001 versus NaCl-vehicle-treated control mice using Student t-tests. Time course of BAL CXCR3+ lymphoid (C) and myeloid (D) infiltrates. Results are expressed as absolute cell counts in the BAL (mean ± SEM). *p < 0.05, ****p < 0.0001 versus LPS-vehicle-treated animals or ### p < 0.001, #### p < 0.0001 versus NaCl-vehicle-treated control mice using Student t-tests. Time course of CXCL12 concentration in the plasma (E) and lung tissue (F). Chemokine concentrations are expressed in ng/ml (plasma) or ng/lung homogenate (mean ± SEM). ****p < 0.0001 versus LPS-vehicle-treated animals or # p < 0.05, ### p < 0.001 versus NaCl-vehicle-treated control mice using Student t-tests. Time course of BAL CXCR4+ lymphoid (G) and BAL CXCR4+ myeloid (H) infiltrates. Results are expressed as absolute counts in the BAL (mean ± SEM). *p < 0.05, **p < 0.01, using Student t-test versus LPS-vehicle-treated animals or ### p < 0.001, #### p < 0.0001 versus NaCl-vehicle-treated control mice using Student t-tests.

Fig 4: 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.