CXCL17 is an allosteric inhibitor of CXCR4 through a mechanism of action involving glycosaminoglycans

Abstract

CXCL17 is a chemokine principally expressed by mucosal tissues, where it facilitates chemotaxis of monocytes, dendritic cells, and macrophages and has antimicrobial properties. CXCL17 is also implicated in the pathology of inflammatory disorders and progression of several cancers, and its expression is increased during viral infections of the lung. However, the exact role of CXCL17 in health and disease requires further investigation, and there is a need for confirmed molecular targets mediating CXCL17 functional responses. Using a range of bioluminescence resonance energy transfer (BRET)-based assays, here we demonstrated that CXCL17 inhibited CXCR4-mediated signaling and ligand binding. Moreover, CXCL17 interacted with neuropillin-1, a VEGFR2 coreceptor. In addition, we found that CXCL17 only inhibited CXCR4 ligand binding in intact cells and demonstrated that this effect was mimicked by known glycosaminoglycan binders, surfen and protamine sulfate. Disruption of putative GAG binding domains in CXCL17 prevented CXCR4 binding. This indicated that CXCL17 inhibited CXCR4 by a mechanism of action that potentially required the presence of a glycosaminoglycan-containing accessory protein. Together, our results revealed that CXCL17 is an endogenous inhibitor of CXCR4 and represents the next step in our understanding of the function of CXCL17 and regulation of CXCR4 signaling.

Fig 1. CXCL17 inhibits constitutive, and ligand-induced CXCR4 signalling.

(A) HEK293 cells transiently transfected with CCR1-10 or CXCR1-3,5 or 6, Gα_i1/Nluc and Venus/Gγ₂ were stimulated with CXCL17, submaximal concentrations of chemokines, CXCL17, or their corresponding chemokine ligands, and their BRET ratios measured. (B) Kinetic analysis of changes in BRET following application of CXCL12, CXCL17 or both CXCL12 and CXCL17 in HEK293 cells transiently-transfected with CXCR4, Gα_i1/Nluc and Venus/Gγ₂. (C) BRET in HEK293 cells transiently transfected with CXCR4, Gα_i1/Nluc and Venus/Gγ₂ and treated with CXCL12 by increasing concentrations of AMD3100 or CXCL17. (D) HEK293G cells stably-expressing the cAMP GloSensor transgene and SNAP/CXCR4 were stimulated with buffer, forskolin, or forskolin and CXCL12, in the absence or presence of CXCL17 or AMD3100. For B, ligand was added following establishment of basal BRET and is indicated on the x-axis as ligand. Points or bars represent mean ± s.e.m. of N=4 (A), N=4 (B), N=4 (C) and N=4 (D) individual experiments. (D) bars represent mean % ± s.e.m. of the forskolin-mediated response of each individual experiment. n.s., non-significant*, p<0.05 and **, p<0.01, indicates a significant difference between the paired groups or group and baseline. Statistical analysis by one-way (D) or two-way (A and B) ANOVA with a Dunnett’s multiple comparisons test.

Fig 2. CXCL17 inhibits constitutive, and ligand-induced CXCR4-β-arrestin2 interactions.

(A) HEK293 cells transiently-transfected with CXCR4/Rluc8 and β-arrestin2/Venus were stimulated at time ligand t1 with HBSS or CXCL12 then at time ligand t2 stimulated again with HBSS or CXCL17. (B) Change in BRET following application of increasing concentrations of CXCL17 to HEK293 cells transiently-transfected with CXCR4/Rluc8 and β-arrestin2/Venus. (C) HEK293 cells transiently-transfected with CXCR4/NLuc and β-arrestin2/Venus were stimulated with CXCL12 in the absence or presence of AMD3100 or CXCL17. (D) HEK293 cells transiently-transfected with CXCR1/Rluc8, CCR5/Rluc8 or β₂-adrenoceptor/Rluc8 and β-arrestin2/Venus were stimulated with CXCL17, CXCL8, CCL5 or isoprenaline respectively. Points or bars represent mean ± s.e.m. of, N=3 (A), N=3, (B), N=3 (D) or N=6, (C). **p<0.01, ***, p<0.01, and ****, p<0.0001 indicates a significant difference between the paired groups or baseline. Statistical analysis by one-way (D) or two-way (A) ANOVA with a Dunnett’s multiple comparisons test.

Fig 3. CXCL17 binding to CXCR4 is context dependent.

Live (A) HEK293 cells or (B) membrane preparations stably-expressing NLuc/CXCR4 were incubated with CXCL12-AF647 and increasing concentrations of AMD3100 or CXCL17. (C) Confocal imaging of HEK293 cells stably-expressing SNAP/CXCR4 under unstimulated conditions or after treatment with CXCL12 or CXCL17. Data in (C) are representative of N=3 individual experiments. Scale bar represents 20 μm. (D) Permeabilised membrane preparations stably-expressing NLuc/CXCR4 were incubated with CXCL12-AF647 in the absence or presence of or CXCL17. White bar (HBSS) is vehicle control. (E) Membrane preparations stably expressing NLuc/CXCR4 were co-incubated with wildtype HEK293 cells and incubated with CXCL12-AF647 and increasing concentrations of AMD3100 or CXCL17. (F) HEK293 cells or (G) membrane preparations stably-expressing NLuc/CXCR4 were incubated with IT1t-BY630/650 and increasing concentrations of IT1t or CXCL17. Points or bars represent mean ± s.e.m. of N=4, (A), N=4, (B), N=4 (D), N=4 (E), N=4, (F) or N=5 (G) individual experiments. ****, p<0.0001 indicates a significant difference between the paired groups or baseline. Statistical analysis by one-way ANOVA with a Dunnett’s multiple comparisons test. For panels B,E,F and G a t-test was performed between the highest concentration of CXCL17 used and fluorescent ligand only, no significant differences were observed p>0.05.

Fig 4. Displacement of the binding of secreted genome-edited CXCL12-HiBiT to SNAP/CXCR4 by CXCL17.

HEK293 cells expressing genome-edited CXCL12-HiBiT, were transiently transfected with SNAP/CXCR4 and incubated in the absence or presence of increasing concentrations of AMD3100,CXCL17 or Surfen. Bars represent basal BRET in the absence of added AlexaFluor488 label. Bars and points represent mean ± s.e.m. of N=6 individual experiments

Fig 5. Binders of glycosaminoglycans mimic the effect of CXCL17.

(A) Sequence of human CXCL17 with putative GAG binding domains highlighted in red. (B-E) effect of GAG binders on ligand binding to Nluc/CXCR4. Live HEK293 cells (B and C) or membrane preparations (D and E) expressing Nluc/CXCR4, were incubated with CXCL12-AF647 (B_E) and increasing concentrations of surfen B and D) or protamine sulfate (C and E), CXCL12 (B and D) or AMD3100 (C and E). Points represent mean ± s.e.m. of N=4,B), N=5, (C), N=4 (D), and N=5 (E) individual experiments.

Fig 6. CXCL17 secretion, membrane interaction, and CXCR4 binding is inhibited by disruption of putative GAG domains.

(A) Sequence of HiBiT tagged wild type CXCL17 or CXCL17 with the putative GAG domains disrupted through alanine substitution. (B) HEK293 cells transfected with CXCL17-HiBiT were treated with CXCL17,surfen or protamine sulfate (PS) with or without heparin sulfate (HS) and luminescence measured. (C) Endpoint luminescence reads of B and figs S9A and S9D, summarising the change in luminescence induced by different treatments in cells transfected with either wildtype CXCL17-HiBiT or gag -ve CXCL17-HiBiT. (D). HEK293 cells were transfected with wildtype CXCL17- or gag -ve CXCL17-HiBiT and SNAP/CXCR4 with BRET measured in the presence and absence of surfen, protamine sulfate, or AMD3100. (E) Raw luminescence output from HEK293 cells transfected with either CXCL17-HiBiT (black bars) or gag -ve CXCL17-HiBiT. Points or bars represent mean ± s.e.m. of N=5 (B), N=5, (C), N=5, (D), N=5, (E) individual experiments. (C-D) *, p<0.05, ****, p<0.0001 calculated by a two-way ANOVA with a Tukey’s multiple comparisons test. (E) ****, p<0.0001 calculated by a Welch’s unpaired t-test (F-G) Proposed mechanisms of CXCL17-mediated inhibition of CXCR4. (F) Inhibition of CXCR4 by CXCL17 ‘bridge’ formation between a closely colocalised GAG-containing proteoglycan and CXCR4. (G) CXCL17-promoted clustering of GAG containing proteoglycans around CXCR4 prevents binding of CXCL12 but allows binding of small molecule inhibitors.

Fig 7. CXCL17 interacts with NRP1 but not VEGFR2.

NanoBRET competition ligand binding in live HEK293 cells expressing (A) NLuc/VEGFR2 or (B) NLuc/NRP1. Cells were incubated with VEGF₁₆₅a-TMR in the absence of other ligands or in the presence of, CXCL17,CXCL12 or VEGF₁₆₅a. Black bar (HBSS) represents vehicle control. (C) HEK293 cells expressing CXCL17-HiBiT were transfected with SNAP/NRP1 and incubated in the absence or presence of surfen or protamine sulfate. Bars represent mean ± s.e.m. of N=4 (A), N=4, (B) or N=5 (C) individual. Data is non-significant unless otherwise specified. n.s., non-significant, *, p<0.05, **, p<0.01, ***, p>0.001, ****, p<0.0001 calculated by one-way ANOVA with Dunnett’s multiple comparisons test.