1. Osteopontin is increased in cystic fibrosis and can skew the functional balance between ELR-positive and ELR-negative CXC-chemokines 
Sandra Jovic, Medya Shikhagaie, Matthias Mörgelin, Jonas S. Erjefält, Sven Kjellström, Arne Egesten 
Journal of Cystic Fibrosis 
Volume 14, Issue 4, Pages (July 2015)
DOI: /j.jcf
Copyright © 2014 European Cystic Fibrosis Society. Terms and Conditions

2. Fig. 1 Enhanced expression of OPN in the airways of CF patients.
Immunohistochemistry to detect OPN was performed on lung tissue obtained from individuals undergoing lung transplantation for end stage CF (A–C; bar=100μm) or donors undergoing surgery for lung cancer (controls) (D–F). Using morphometric analysis, significantly less OPN was detected intra-epithelially of control lung tissue compared to CF lung tissue (G). Using an ELISA, OPN was quantified in sputum of CF patients, at a mean concentration of 2735pg/mL (range: 892–8170pg/mL; n=26) and in induced sputum from healthy controls, at a mean concentration of 53pg/mL (range 45–63pg/mL; n=3) (H). Mann–Whitney's U-test for unpaired samples was used for comparison between the groups. A P-value of less than 0.05 was considered significant (*P<0.05; ***P<0.001).
Journal of Cystic Fibrosis  , DOI: ( /j.jcf )
Copyright © 2014 European Cystic Fibrosis Society. Terms and Conditions

3. Fig. 2 Polymers and fragments of OPN in CF sputum.
Western blot was performed to detect OPN in CF sputum and in induced sputum from healthy controls. Full length OPN was detected at approximately 65kDa (asterisk). In induced sputum from healthy controls (individuals 1–3), weak presence of OPN was seen. However, in CF sputum (individuals 4 and 5) strong presence of full length monomeric OPN (asterisk), numerous OPN-fragments and polymeric OPN was seen. In vitro, elastase of P. aeruginosa (PE) and neutrophil elastase (NE) released fragments of similar sizes as detected in CF sputum (arrows and arrowhead respectively) (A). The OPN fragment generated by PE (VSS) is derived from the mid-portion of OPN as indicated in blue, and the NE generated OPN fragment (LNA) is derived from the N-terminal portion of OPN as indicated in yellow (B). The biophysical properties of OPN and the fragments are shown (C).
Journal of Cystic Fibrosis  , DOI: ( /j.jcf )
Copyright © 2014 European Cystic Fibrosis Society. Terms and Conditions

4. Fig. 3 Binding of OPN to CXC-chemokines.
Surface plasmon resonance analysis of OPN binding to CXC-chemokines. The strongest binding was measured between MIG/CXCL9 and OPN (KD=113nM) followed by I-TAC/CXCL11 and OPN (KD=1842nM) while IP-10/CXCL10 had a weaker binding to OPN (KD=15,060nM). Interestingly, no binding was observed between GRO-β/CXCL2, GCP-2/CXCL6, IL-8/CXCL8 and OPN (A). The enzyme-linked binding-assay confirmed the results obtained using surface plasmon resonance. However, a low binding between GRO-β/CXCL2, GCP-2/CXCL6, IL-8/CXCL8 and OPN was observed (B). Scanning electron microscopy (SEM) was performed to investigate the morphology of P. aeruginosa after incubation in buffer alone, with MIG/CXCL9 (1μM) conjugated with 5nm colloidal gold, or with MIG/CXCL9 pre-incubated with OPN (1μM) conjugated with 10nm colloidal gold (C). One representative out of three separate experiments is shown.
Journal of Cystic Fibrosis  , DOI: ( /j.jcf )
Copyright © 2014 European Cystic Fibrosis Society. Terms and Conditions

5. Fig. 3 Binding of OPN to CXC-chemokines.
Surface plasmon resonance analysis of OPN binding to CXC-chemokines. The strongest binding was measured between MIG/CXCL9 and OPN (KD=113nM) followed by I-TAC/CXCL11 and OPN (KD=1842nM) while IP-10/CXCL10 had a weaker binding to OPN (KD=15,060nM). Interestingly, no binding was observed between GRO-β/CXCL2, GCP-2/CXCL6, IL-8/CXCL8 and OPN (A). The enzyme-linked binding-assay confirmed the results obtained using surface plasmon resonance. However, a low binding between GRO-β/CXCL2, GCP-2/CXCL6, IL-8/CXCL8 and OPN was observed (B). Scanning electron microscopy (SEM) was performed to investigate the morphology of P. aeruginosa after incubation in buffer alone, with MIG/CXCL9 (1μM) conjugated with 5nm colloidal gold, or with MIG/CXCL9 pre-incubated with OPN (1μM) conjugated with 10nm colloidal gold (C). One representative out of three separate experiments is shown.
Journal of Cystic Fibrosis  , DOI: ( /j.jcf )
Copyright © 2014 European Cystic Fibrosis Society. Terms and Conditions

6. Fig. 4
OPN inhibits the antibacterial activity of ELR-negative chemokines.
MIG/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11 (1μM) alone showed high bacterial killing. After pre-incubation with OPN at equimolar concentrations or at a ration of 1:0.1, the antibacterial activities of the chemokines were significantly reduced (A). The two elastase derived OPN-fragments, VSS and LNA did not reduce the bactericidal activity of MIG/CXCL9, IP-10/CXCL10 and I-TAC/CXCL11 to the same extent as the full-length protein did (B and C respectively). Mann–Whitney's U-test for unpaired samples was used for comparison between the groups. A P-value of less than 0.05 was considered significant (*P<0.05; **P<0.01; ***P<0.001).
Journal of Cystic Fibrosis  , DOI: ( /j.jcf )
Copyright © 2014 European Cystic Fibrosis Society. Terms and Conditions

7. Fig. 5
OPN interferes with activation of CXCR3-bearing cells but not neutrophils.
MIG/CXCL9 IP-10/CXCL10, and I-TAC/CXCL11 (100nM) alone or pre-incubated with OPN (100nM) were investigated for their calcium-mobilizing properties in CXCR3-transfected cells (A). Likewise, GRO-β/CXCL2, GCP-2/CXCL6, and IL-8/CXCL8 alone and pre-incubated with OPN were investigated for intracellular calcium-mobilization in purified neutrophils (B). One representative and the mean and SD of three separate experiments respectively are shown. Unpaired t-test followed by Welch's correction was used for comparison between the groups. A P-value of less than 0.05 was considered significant (*P<0.05).
Journal of Cystic Fibrosis  , DOI: ( /j.jcf )
Copyright © 2014 European Cystic Fibrosis Society. Terms and Conditions