key: cord-0273345-b7th1ylz
authors: White, Carl W.; Kilpatrick, Laura E.; Dale, Natasha; Abhayawardana, Rekhati S.; Dekkers, Sebastian; Stocks, Michael J; Pfleger, Kevin D. G.; Hill, Stephen J.
title: CXCL17 is an endogenous inhibitor of CXCR4 via a novel mechanism of action
date: 2021-07-05
journal: bioRxiv
DOI: 10.1101/2021.07.05.451109
sha: 666d5443fd9cfa41b9c2daed2a4f7bd44904617c
doc_id: 273345
cord_uid: b7th1ylz

CXCL17 is the most recently described chemokine. It is 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, as well as being highly upregulated during viral infections of the lung. However, the exact role of CXCL17 in health and disease is largely unknown, mainly due to a lack of known molecular targets mediating CXCL17 functional responses. Using a range of bioluminescence resonance energy transfer (BRET) based assays, here we demonstrate that CXCL17 inhibits CXCR4-mediated signalling and ligand binding. Moreover, CXCL17 interacts with neuropillin-1, a VEGFR2 co-receptor. Additionally, we find CXCL17 only inhibits CXCR4 ligand binding in intact cells and demonstrate that this effect is mimicked by known glycosaminoglycan binders, surfen and protamine sulfate. This indicates that CXCL17 inhibits CXCR4 by a unique mechanism of action that potentially requires the presence of a glycosaminoglycan containing accessory protein. Altogether, our results reveal that CXCL17 is an endogenous inhibitor of CXCR4 and represents an important discovery in our understanding of the (patho) physiological functions of CXCL17 and regulation of CXCR4 signalling.

Chemokines are a large family of small, secreted cytokines that play a central role in the migration of cells. Chemokines are widely expressed throughout the body and facilitate both homeostatic as well as inflammatory immune responses. In addition, chemokines mediate numerous other physiological functions including angiogenesis and organogenesis as well as participate in pathophysiological processes such as cancer progression, autoimmune disorders, and aberrant inflammation 1 . Chemokines induce cellular responses by binding and activating G protein-coupled receptors (GPCRs). This results in the activation of heterotrimeric G proteins followed by downstream signalling, with subsequent βarrestin recruitment to the chemokine receptor resulting in signal termination via receptor internalisation and desensitization 2 .

CXCL17 is the most recently described chemokine. It is constitutively expressed at high levels in mucosal sites throughout the body and is thought to be involved in the innate immune response via recruitment of monocytes, dendritic cells and macrophages 3, 4 . In addition, CXCL17 has antimicrobial properties at high concentrations 5 and modulates angiogenesis 4, 6 . CXCL17 has also been implicated in several pathologies including breast 7 and hepatocellular cancers 8 , as well as inflammatory lung pathologies such as idiopathic pulmonary fibrosis 5 , asthma 9 , influenza 10 and SARS-CoV-2 11 infection.

Despite mediating a wide range of important functional responses, the receptor(s) for CXCL17 are still unknown. Thus, it is vital to determine the molecular targets of CXCL17 not only to fully understand the normal function of CXCL17 but to allow targeting of its pathophysiological effects.

Previous work implicated GPR35 as the receptor for CXCL17 12 , however subsequent studies 13, 14 have been unable to reproduce this finding. Despite this, G protein-coupled receptors likely play a role in CXCL17-mediated signalling. Indeed, CXCL17-mediated calcium flux 12 , inhibition of cAMP accumulation 15 and pertussis toxin sensitive signalling 4, 16 have all been observed. Very recently, we have found CXCL17 is able to activate the atypical chemokine receptor ACKR3 as well as interact with glycosaminoglycans (manuscript in preparation). Since ACKR3 does not activate G protein-mediated signalling 17 , it is unlikely to be the only receptor for CXCL17. Notably ACKR3 interacts closely with the chemokine receptor CXCR4, through formation of ACKR3-CXCR4 heteromers as well as binding and scavenging CXCL12, the cognate ligand for CXCR4 18 .

CXCR4 is a prototypical chemokine receptor that facilitates numerous physiological processes including organogenesis, hematopoiesis, and immune responses via binding of CXCL12. Moreover, CXCR4 signalling is involved in multiple diseases including various cancers, autoimmune disorders, aberrant inflammation and HIV-1 infection 19 . Therefore, the development of inhibitors of CXCR4 is an area of active research 20 . Notably, CXCL12 and CXCR4 are constitutively-expressed at high levels widely throughout the body and several mechanisms exist to regulate CXCR4 signalling. These include: receptor oligomerisation; scavenging of CXCL12 by ACKR3 to regulate ligand localization and abundance; post-translational modifications; and proteolytic degradation of CXCL12 19 . Moreover, positively charged domains within CXCL12 mediate interactions with glycosaminoglycans that facilitate CXCL12 oligomerisation, prevent proteolytic degradation and help establish chemotactic gradients 21 . Finally, several endogenous inhibitors of CXCR4 have now been reported including CXCL14 22 , the endogenous cationic antimicrobial peptide cathelicidin LL37 23 , as well as EPI-X4, an endogenous peptide fragment of serum albumin 24 .

In this study, across a panel of chemokine receptors we find no agonist-like responses mediated by CXCL17. Instead we show that CXCL17 inhibits CXCR4-mediated signalling as well as ligand binding in intact live cells. CXCL17 is therefore an endogenous inhibitor of CXCR4 and we discuss the potential physiological implications of this interaction. Notably, CXCL17 has no effect on binding of CXCL12 or a small molecule antagonist to CXCR4 in dissociated membrane preparations. This suggests that CXCL17 inhibits CXCR4 by a unique mechanism of action which we propose involves an accessory protein containing glycosaminoglycans.

In initial confirmatory experiments we sought to rule out GPR35 as a target for CXCL17 using bioluminescence resonance energy transfer (BRET) assays. In HEK293 cells transiently-transfected with GPR35/NLuc and β-arrestin2/Venus (Supplementary Figure 1a) conformational change of the G protein complex (except for CCR9 for which no response was obtained with the positive control ligand) however CXCL17 (30 nM, sufficient to induce signalling 6 ) produced no comparable or observable change in the BRET ratio. Taken together these data confirm GPR35 is not a direct target of CXCL17.

Targets of CXCL17 are likely to be Gαi coupled G protein-coupled receptors due to reports of pertussis toxin sensitive signalling 4, 16 . In HEK293 cells transfected with Gαi1/NLuc, Venus/Gγ2 and an individual chemokine receptor CCR1-10 or CXCR1-3, 5 or 6 (without co-expression of GPR35), endogenous chemokine ligands resulted in a decrease in the BRET ratio suggestive of receptor activation, however, no such change was observed using a high concentration of CXCL17 (300 nM) either in the absence or presence of endogenous chemokine ligand (Figure 1a, n=4) . Surprisingly, while CXCL17 did not induce an agonist-like response, we found that in HEK293 cells co-expressing CXCR4, Gαi1/Nluc and Venus/Gγ2, CXCL17 (300 nM) resulted in an increase in the BRET ratio as well as inhibited responses mediated by CXCL12 (1 nM, Figure 1b ) in a concentration-dependent manner (Figure 1c ; CXCL12 0.1 nM, pIC50 = 6.93 ± 0.28, n=4) albeit with a lower potency than the small molecule CXCR4 antagonist AMD3100 (pIC50 = 7.52 ± 0.03, n=4). CXCL17 had similar inhibitory effects in Cos-7 cells transiently transfected with CXCR4, Gαi1/NLuc and Venus/Gγ2 , indicating these effects where not cell type dependent (Supplementary Figure 3) . To investigate the effect of CXCL17 on downstream CXCR4 signalling we stably-transfected SNAP/CXCR4 into HEK293G cells that express the GloSensor cAMP biosensor. In this system CXCL17 and the small molecule CXCR4 antagonist AMD3100 inhibited the CXCL12-mediated decrease in forskolin-induced cAMP accumulation (Figure 1d , p<0.01, n=4, one way ANOVA with Dunnett's multiple comparisons test). Interestingly, in the Gαi1/NLuc and Venus/Gγ2 activation assay using a constitutively-active CXCR4 mutant (N119S), we observed an increase in the magnitude of inhibition (compared to at wildtype CXCR4) mediated by a submaximal concentration of CXCL17 (100 nM, p<0.05, WT CXCR4 versus N119S CXCR4, n=3, two-tailed unpaired t-test), whereas AMD3100 (1 µM) appeared to mediate a small decrease in BRET indicative of receptor activation and is in keeping with reports of AMD3100 being a partial agonist for constitutively active CXCR4 25 (Figure 1e) . Finally, N-terminal truncation of chemokines can drastically modulate function, indeed an increase in CXCL17 potency has been observed following N-terminal truncation 6 . 24Leu-CXCL17 has been used in most reports and is the canonical version of CXCL17 we have used here.

However, in a direct comparison we observed no difference in the ability of 24LeuCXCL17 or a commercially available truncated version 22Ser-CXCL7 to inhibit constitutive CXCR4 mediated changes in Gαi1/NLuc-Venus/Gγ2 conformation in HEK293 cells (Figure 1f , p>0.05, n=3). Similarly, 22Ser-CXCL17 (300 nM) still inhibited the decrease in BRET mediated by CXCL12 (1 nM, Figure 1f , p<0.05, n=3). Altogether these results suggest CXCL17 selectively inhibits CXCR4 signalling and has a different mechanism of action compared to small molecule antagonists.

To confirm the inhibitory effects of CXCL17 we next investigated its effect on recruitment of β-arrestin-2 to the CXCR4. In HEK293 cells transiently transfected with CXCR4/Rluc8 and β-arrestin2/Venus, application of CXCL17 (100 nM, Figure 2a , n=3) resulted in a decrease in the basal BRET ratio as well as inhibition of CXCL12 (100 nM) mediated increases in BRET, thus indicating CXCL17 inhibits both constitutive and ligand-induced CXCR4 activity. The decrease in the basal BRET ratio following the application of CXCL17 was concentration-dependent (Figure 2b, pIC50 = 7.24 ± 0.08, n= 3) and in HEK293 cells transfected with CXCR4/NLuc and β-arrestin2/Venus ( Figure 2c ) both CXCL17 (pIC50 = 6.85 ± 0.30, n=6) and AMD3100 (pIC50= 7.79 ± 0.14, n=6) inhibited the increase in BRET mediated by CXCL12 (10 nM) in a concentration-dependent manner. To further test the specificity of CXCL17 for CXCR4, we used HEK293 cells transfected with CXCR1/Rluc8, CCR5/Rluc8 or β2adrenoceptor/Rluc8 (β2-AR) and β-arrestin2/Venus. Each configuration showed an increase in BRET following application of a prototypical agonist but no modulation of the BRET ratio following application of CXCL17 (300 nM, Figure 2d , n=3).

Initial observations suggested that CXCL17 had a unique mechanism of action/binding compared to known antagonists. To further examine binding of CXCL17 to CXCR4 we used a NanoBRET ligand binding approach. In live HEK293 cells stably-expressing exogenous CXCR4 tagged on the N-terminus with NanoLuc (NLuc/CXCR4), both AMD3100 and CXCL17 competed with CXCL12-AF647 (12.5 nM, Figure 3a , pKi = 7.99 ± 0.16 and pKi = 6.25 ± 0.19 respectively, n=4) for NLuc/CXCR4 binding in a concentration-dependent manner. Recent studies have demonstrated the oligomeric state of CXCR4 is expression dependent and that oligomer formation and signalling can be disrupted by ligands such as IT1t that bind allosterically 26, 27 . However, when binding experiments were performed in live HEK293 or HeLa cells CRISPR/Cas9 genome-edited to express NLuc/CXCR4 under endogenous promotion and therefore with native levels of CXCR4 expression (Supplementary Figure 4) , AMD3100 (pKi = 8.01 ± 0.13 and pKi = 8.30 ± 0.08 respectively, n=4) and CXCL17 (pKi = 6.14 ± 0.30 and pKi = 6.15 ± 0.27 respectively, n=4) inhibited CXCL12-AF647 (12.5 nM) binding. These results rule out that this effect was specific to over-expressed CXCR4 receptors, potentially existing predominantly as CXCR4 oligomers, and further demonstrating inhibition was independent of cell type. Indeed, supporting a unique binding mode, CXCL17 did not compete with CXCL12-AF647 binding to NLuc/CXCR4 in membrane preparations from HEK293 cells stably-overexpressing the receptor (Figure 3b Finally, on close inspection of the competition ligand binding curves (Figures 3a-b) we noted a steep slope >1 suggestive of non-competitive and/or allosteric interactions and therefore hypothesised CXCL17-mediated ligand displacement would be probe-dependent. To this end, the CXCR4 antagonist IT1t but not CXCL17 competed with binding of IT1t-BY630/650 (a fluorescent derivative of IT1t, pKd = 7.25 ± 0.14, n=5, Supplementary Figure 6 ) to NLuc/CXCR4 expressed in live HEK293 cells or to NLuc/CXCR4 in membrane preparations (Figure 3f -g, pKi = 7.89 ± 0.06 and pKi = 7.92 ± 0.06 for IT1t in cells and membranes respectively, n=4-5). Since the binding site of IT1t only partially overlaps with CXCL12 28 , these results further indicate that CXCL17 binds by a unique mechanism/site compared to the endogenous agonist CXCL12 and other known CXCR4 antagonists.

In our initial experiments we also noted that CXCL17 inhibits basal/constitutive CXCR4 signalling, suggesting inverse agonist activity. However, CXCL12 is endogenously expressed in HEK293 cells at levels sufficient to initiate signalling 29 . To investigate if CXCL17 was disrupting signalling mediated by endogenous CXCL12, we took advantage of HEK293 cells engineered using CRISPR/Cas9 genomeediting to express CXCL12 tagged with a small 11 amino acid self-complementing fragment of NLuc (HiBiT). When the cells were transfected with exogenous SNAP/CXCR4, AMD3100 and CXCL17 both produced a concentration-dependent decrease in BRET ( Figure 4 , pIC50 = 7.10 ± 0.08 and 6.27 ± 0.16 respectively, n=6), generated between CXCL12-HiBiT complemented with LgBiT and SNAP/CXCR4 labelled with cell-impermeant AlexaFluor488. These data indicate that inhibition of constitutive CXCR4 signalling is likely due to blocking endogenous CXCL12 binding to CXCR4.

Chemokines in addition to membrane receptors bind GAGs via basic domains (putatively BBxB) that result in chemokine accumulation at the cell surface and facilitates oligomerisation as well as prevents proteolytic degradation. CXCL17 has an overall positive net charge of +18 at pH 7.4 and contains multiple highly conserved (Supplementary Figure 7) putative GAG domains ( Figure 5a ) indicating an ability to bind GAGs potentially with high affinity. As generation of membrane preparations undoubtedly disrupts the extracellular matrix, we therefore hypothesised that CXCL17 required the presence of (unperturbed) membrane-bound glycosaminoglycans to inhibit CXCR4. To test this, we determined whether the effect of CXCL17 could be mimicked by known GAG binders. Both the small molecule GAG inhibitor surfen and the heparin antidote protamine sulfate (previously shown to inhibit CXCR4 signalling 30 Figure 8a) . Next, we used exogenous soluble heparan sulphate to pre-occupy the GAG binding sites of CXCL17 and therefore reduce its capacity to bind to endogenous GAG in the extracellular matrix. We observed that while heparan sulfate (30 µg/ml) treatment had no effect on the binding of CXCL12-AF647 (12.5 nM) to NLuc/CXCR4 in live HEK293 cells (Supplementary Figure 8b) , preincubation of CXCL17 (300 nM) with heparan sulfate (30 µg/ml) reduced the ability of CXCL17 to cause displacement of CXCL12-AF647 binding to NLuc/CXCR4 (p<0.01, n=5) whereas heparan sulfate had no effect on AMD3100 (1 µM). Finally, we investigated if inhibition of CXCR4 ligand binding was a general property of high affinity GAG binders. Using a high concentration of CXCL4 (1 µM, a.k.a. platelet factor 4, GAG affinity ~ 30 pM 31 ) we did not observe inhibition of CXCL12-AF647 (12.5 nM) binding to NLuc/CXCR4 in live HEK293 Figure 8c) . These results suggest GAGs are involved in the CXCL17 mediated inhibition and binding of CXCR4, but that not all known GAG binders can inhibit binding of CXCL12 to CXCR4.

While GAGs are important regulators of chemokine function in vivo, it is hypothesised that they are not strictly required for receptor binding. Indeed, dual GAG-receptor binding appears sterically unlikely for most chemokines 32 . In contrast, GAG binding motifs are required for ligand binding to some receptor tyrosine kinases 33 . Indeed, the vascular endothelial growth factor receptor 2 (VEGFR2) and its co-receptor neuropilin-1 NRP1 both bind full length VEGF-A, however alternatively spliced isoforms of VEGF that lack a GAG binding motif do not bind to NRP1 34, 35 Taken together, these results further support the involvement of GAG binding motifs in the binding mechanism of CXCL17 and suggests a direct interaction with VEGF signalling pathways.

CXCL17 was first described in the literature as having chemotactic properties for dendritic cells and monocytes 4 , as well as being correlated with VEGF expression 37 . Since then, our knowledge of CXCL17 in both health and disease has expanded to include functions as an important innate immune factor at mucosal barriers, regulation of angiogenesis, and involvement in tumorigenesis (for review see 38 ) . However, our current understanding is limited by a lack of bonafide molecular targets via which CXCL17 binds and elicits its function. Previously, GPR35, an orphan G protein-coupled receptor, was reported to be a receptor for CXCL17 12 . However, multiple investigations 13,14 , including those presented here have failed to observe similar CXCL17-GPR35 interactions.

Here we report an inhibitory interaction between CXCL17 and CXCR4 across multiple assays, cell lines and contexts. These results contrast with previous studies that found no effect on CXCR4 signalling or ligand binding by CXCL17 6,12 . However, this is unsurprising given the fact that the focus of the previous investigations was on agonist-induced signalling (unlike the antagonistic responses seen here), and that previous ligand binding studies used membrane preparations which, as demonstrated in the current study, do not permit CXCL17 binding to CXCR4. In vivo, endogenous CXCR4 inhibitors may represent an important regulatory mechanism to prevent excessive receptor activation. Notably CXCL17 expression can reach ~ 60 ng/ml in vitro while serum CXCL17 concentrations can reach ~ 5 ng/ml during influenza infection 10 . Since CXCL17 expression is highly localised to secretory cells in mucosal tissue 39 it is plausible that CXCL17 expression reaches concentrations sufficient to inhibit CXCR4.

When compared to AMD3100, which has been reported to be a weak partial CXCR4 agonist 25 , and the inverse agonist IT1t 40 , CXCL17 has a unique mechanism by which inhibition of CXCR4 is achieved.

Here the most striking evidence for this is the inability of CXCL17 to inhibit CXCL12-AF647 binding to CXCR4 in membrane preparations. We initially considered that a lack of CXCL17-mediated effects in membranes preparations was due to partitioning or internalisation of CXCR4 in cells. However, limiting ligand binding to the plasma membrane using HiBiT-tagged CXCR4 or cell permeabilization did not support such a mechanism. We also found that CXCL17 did not displace a fluorescently-labeled derivative of IT1t from Nluc/CXCR4 in whole live cells or membrane preparations which could suggest that the formation of CXCL12-AF647-CXCL17 interactions (heteromerization) contributed to CXCL17 inhibition of CXCR4. However, such an interaction would also be expected to occur in membrane preparations when co-incubated with live cells.

In our efforts to delineate the mechanism of action of CXCL17 we noted a steep slope >1 of CXCL17mediated displacement of CXCL12-AF647 in the NanoBRET competition ligand binding curves which is indicative of an allosteric and/or non-competitive interaction. Furthermore, since CXCL17 had no effect in membrane preparations this suggested the involvement of a site that was lost once intact cells were disrupted and pointed to the involvement of an accessory protein. Here we present several lines of evidence that this potential accessory protein contains glycosaminoglycans: 1) CXCL17 contains multiple highly conserved putative GAG binding domains and preparation of purified cell membranes certainly disrupts the native structure, function and/or potentially presence of GAGs 41 . 2) In support of this, the effect of CXCL17 was mimicked by known GAG binders, surfen and protamine sulphate.

Further evidence from the present study for the involvement of GAGs are: a) heparan sulfate reduced CXCL17-mediated inhibition of CXCL12-AF647 NLuc/CXCR4 binding presumably via blockade of CXCL17 GAG binding domains and b) CXCL17 inhibited binding of VEGF165a-TMR binding to NLuc/NRP1 but not NLuc/VEGFR2. GAG binding domains are strictly required for VEGF165a binding to NRP but not for binding to VEGFR2 35 . Notably there does appear to be a level of specificity for CXCL17-mediated inhibition of CXCR4 since the high affinity GAG binder CXCL4 31 did not inhibit CXCR4, and CXCL17 does not broadly inhibit or activate other chemokine receptors. While yet to be determined, such specificity may be encoded through a dependence on the composition of the GAG side chains (i.e. heparan sulfate versus chondroitin sulfate) for CXCL17 binding, the need for CXCL17 to contain both GAG and CXCR4 binding domains, or inhibition of CXCR4 may depend on interactions with a specific membrane proteoglycan to bring specific GAG binding domains in close proximity with A plausible explanation for these results could be that CXCL17 participates in the release of an endogenous factor that subsequently binds and inhibits CXCR4. Indeed, competitive chemokine binding for GAGs is known to induce cooperative signalling due to the 'release' of GAG bound chemokines subsequently activating a secondary receptor 43 . However, CXCL17 had no effect on CXCL12-CXCR4 interactions when membrane preparations expressing NLuc/CXCR4 were coincubated with live intact wildtype HEK293 cells, making such an effect unlikely. In contrast, when considering the mechanism of CXCL17 inhibition of CXCR4, these results principally support transinteractions that are between CXCR4 and a proteoglycan/GAG on the same cell rather than cis interactions between receptors and GAGs on different cells. Thus, the simple presence of GAGs is not sufficient for CXCL17-mediated inhibition of CXCR4 and indicates that they also need to be in the correct orientation and/or close proximity.

It is notable that CXCR4 can form complexes with membrane proteoglycans 44, 45 and taken together this leads us to propose two potential modes of inhibition by CXCL17: 1) A 'bridge formation' where CXCL17 simultaneously binds to CXCR4 and a GAG in close proximity to the receptor (Figure 5f ) or 2) a result of clustering of GAGs induced by CXCL17 in the vicinity of CXCR4 causing steric hindrance and reduced access of CXCL12 to the receptor (Figure 5g ). In the first mechanism, elements of the GAG sidechain must contribute to the binding of CXCL17 to CXCR4. While we recognise that for most chemokines simultaneous binding to GAGs and chemokine receptors is unlikely due to the overlap of chemokine binding sites and/or steric hinderance 32 , potential exceptions do exist; for example, for CCL21 and CXCL12γ that have extended C-terminal tails enriched in basic residues 21 . As the putative GAG binding domains of CXCL17 are positioned toward its C-terminus, similar to the situation with CCL21 and CXCL12γ, dual receptor-GAG binding may be structurally permitted. In support of option (2) , chemokines are known to induce clustering of GAGs/proteoglycans 21 . Therefore, CXCL17mediated clustering and stiffening of membrane proteoglycan complexes around CXCR4 could hinder the access of CXCL12 to the extracellular domains of CXCR4. This may also explain why CXCL17 did not interfere with the binding of a small molecule fluorescent derivative of IT1t, which binds within a transmembrane pocket of CXCR4 28 and may more readily penetrate through clustered proteoglycans.

While the exact mechanism of CXCL17-mediated inhibition requires further investigation, it is worth noting that the extracellular domains of CXCR4 are highly acidic and inhibition by highly basic peptides is a known phenomenon 46 . Positively charged CXCR4 inhibitors include the synthetic peptides TT22 CXCL17 has been reported to facilitate angiogenesis principally via secretion of pro-angiogenic factors such as VEGF-A and b-FGF 6,37 . Here binding of CXCL17 to NRP1 represents a direct interaction with the prototypical VEGF-A angiogenic pathway. NRP1 acts as a co-receptor that selectively potentiates VEGFR2 signaling, a key driver of angiogenesis, and has been reported to directly promote angiogenesis 34 . While we did not directly assess the effect on VEGF-A mediated signaling or angiogenesis, CXCL17-mediated angiogenesis via interactions with NRP1 is plausible and should be examined further. Furthermore, the potential for interplay between chemokines and receptor tyrosine kinases (RTKs) due to potential overlapping glycosaminoglycan binding is an intriguing finding, particularly since some RTKs require formation of an obligate RTK-GAG complex for ligand binding, for example FGF-2 binding to FGFR 48 .

In summary, we demonstrate that CXCL17 inhibits CXCR4 and does so potentially via a glycosaminoglycan-containing accessory protein. These data suggest a unique mechanism of action that could be targeted to therapeutically inhibit CXCR4 in a cell type specific manner. Additionally, we confirm that GPR35 does not play a role in the function of CXCL17 and furthermore demonstrate CXCL17 has the potential to modulate VEGF signalling pathways via direct interactions with the VEGFR2 co-receptor NRP1. Together, the identification of CXCR4 and NRP1 as new molecular targets is a substantial contribution to our understanding of CXCL17 and may provide important insights into the patho/physiological roles of CXCL17.

AMD3100 was purchased from Selleckchem (USA) or Sigma-Aldrich (Australia). Unlabeled chemokine ligands were purchased from Preprotech (USA) except for 24Leu-CXCL17 which was purchased from R&D Systems and 22Ser-CXCL17 which was purchased from Sapphire Bioscience AMD3100 was dissolved in water, unlabeled chemokines and CXCL12-AF647 were dissolved as per the manufacturer's instructions. VEGF165a was dissolved in phosphate buffered saline (PBS) containing 0.1% bovine serum albumin (BSA; Sigma Aldrich). IT1t and Zaprinast were dissolved in DMSO to a concentration of 10 mM. All further dilutions were performed in assay buffer containing 0.1% BSA.

Expression cDNA constructs encoding untagged chemokine receptors, β2-adrenoceptor or GPR35 were purchased from the cDNA Resource Center. To generate the pcDNA3. On the day of assay, cells were washed and incubated with Hanks buffer for 1 hour at 37 °C before furimazine (10 µM) was added. Cells were incubated for a further 5 mins at 37 °C before real-time BRET measurements were taken at 37 °C using a LUMIstar as described above. Following 

Genome-edited cells expressing NLuc/CXCR4 were seeded into poly-D-lysine coated white flat bottom 96 well plates at 30,000 cells/well and incubated for 24h at 37°C/5% CO2. On the day of the assay, cells were washed and incubated with pre-warmed HBSS supplemented with 0.1% BSA and incubated with increasing concentrations of IT1t-BY630/650 in the absence or presence of IT1t (10 µM) for 1 hour at 37°C. Furimazine (10 µM) was added, and plates equilibrated for 5 minutes at room temperature before sequential filtered light emissions were taken using a PHERAStar FS plate reader using 460nm (80nm bandpass) and >610nm (longpass) filters. BRET ratios were calculated by dividing the 610nm emission (acceptor) by the 460nm emission (donor).

HEK293 cells stably expressing HiBiT/CXCR4 were seeded into poly-D-lysine coated white flat bottom 96 well plates at 30,000 cells/well and incubated for 24h at 37°C/5% CO2. On the day of the assay, cells were washed and incubated with pre-warmed HBSS supplemented with 0.1% BSA. A concentration-response curve was generated by incubating cells with CXCL17 (10 pM -1 uM) for 60 minutes at 37°C. Following ligand incubation, furimazine (10 μM) and purified LgBiT (10 nM) were added and plates incubated for 5 minutes before total light emissions were continuously measured using a PHERAStar FS plate reader with the concentration-response curves representing the luminescence after 30 minutes.

HEK293G cells stably expressing SNAP/CXCR4 were seeded into poly-D-lysine coated 8-well plates at 10,000 cells/well and incubated for 24h at 37°C/5% CO2. On the day of the assay, cells were incubated with 0.5 µM membrane impermeant SNAP-tag AF488 for 30 minutes at 37°C/5% CO2 prepared in serum-free DMEM. Cells were then washed three times with HBSS/0.1% BSA and incubated with CXCL12 (100 nM), CXCL17 (300 nM) or vehicle (HBSS/0.1% BSA) for 1 hour in the dark at 37°C.

Cells were imaged live at 37°C using a Zeiss LSM710 fitted with a 63x Plan Apochromat oil objective (1.4NA) using a Argon488 (AlexaFluor488; 496-574nm band pass; 2% power) with a 488/561/633 beamsplitter using a pinhole diameter of 1 Airy unit. All images were taken at 1024x1024 pixels per frame with 8 averages (zoom 1).

HEK293 cells expressing genome-edited CXCL12-HiBiT were seeded in 6 well plates at 300,000 cells per well and incubated for 24h at 37°C/5% CO2. Cells were then transfected with 500 ng/well BRET ratios were calculated by dividing the 535 nm emission (acceptor) by the 475 nm emission (donor).

Conservation analysis was performed by WebLogo 55 using the multiple sequence alignment of fortyfive 119 amino acid CXCL17 orthologues obtained from UniProt 56 (Supplementary Table 1 ).

Due to differences in plate reader sensitivity and expression differences between cell lines/assays, raw BRET ratios cannot be compared as a measure of BRET efficacy between figures as optimised plate reader emission gains were used to ensure sufficient sensitivity and/or measurements acquired did not saturate the detector. In general, BRET ratios were calculated by dividing the acceptor emission by the donor emission. Calculation of baseline-corrected BRET ratios or luminescence values are described in the methods for each assay configuration. Where results reported are taken from kinetic reads, points or bars are the maximum ligand-induced change in the BRET ratio.

Prism 8 software was used to analyse ligand-binding curves. For NanoBRET receptor-ligand saturation binding assays, total and non-specific saturation binding curves were simultaneously fitted using the following equation: 

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Figure Legends Figure 1: CXCL17 inhibits constitutive, and ligand-induced CXCR4 signalling. (a) HEK293 cells transiently transfected with CCR1-10 or CXCR1-3,5 or 6, Gαi1/Nluc and Venus/Gγ2 were stimulated with CXCL17 (300 nM

CCL13 (1 nM), CCL22 (0.3 nM), CCL4 (30 nM), CCL20 (3 nM), CCL19 (1 nM), CCL1 (10 nM), CCL25 (10 nM), CCL27 (10 nM), CXCL8 (0.3 nM)

300 nM) and the corresponding chemokine ligand (grey bars). (b) Kinetic analysis of changes in BRET following application of CXCL12 (1 nM, black squares), CXCL17 (300 nM, white circles) or both CXCL12 and CXCL17 (1 nM and 300 nM respectively, white squares) in HEK293 cells transiently-transfected with CXCR4, Gαi1/Nluc and Venus/Gγ2. (c) Inhibition of the change in BRET

HEK293 cells transiently transfected with CXCR4, Gαi1/Nluc and Venus/Gγ2 by increasing concentrations (1 nM -1 µM) of AMD3100 (black squares) or CXCL17 (white circles). (d)

(e) HEK293 cells transiently-transfected with Gαi1/Nluc, Venus/Gγ2 and wildtype (WT) CXCR4 or the CXCR4 N119S constitutively-active mutant were stimulated with CXCL12 (10 nM, black bars), CXCL17 (100 nM, white bars) or AMD3100 (10 µM, grey bars). (f) In parallel, HEK293 cells transiently-transfected with CXCR4, Gαi1/Nluc and Venus/Gγ2 were stimulated with canonical CXCL17 (24Leu-CXCL17, 300 nM), a truncated version of CXCL17 (22Ser-CXCL17, 300 nM), CXCL12 (1 nM), or 22Ser-CXCL17 (300 nM) and CXCL12 (1 nM). For b, ligand was added following establishment of basal BRET and is indicated on the x-axis as ligand. Corrected BRET was calculated as described in Methods. Points or bars represent mean ± s.e.m. of three (e and f) or four (ad) individual experiments performed in duplicate or triplicate. (d) bars represent mean % ± s.e.m. of the forskolin-mediated response of each individual experiment. *, p<0.05 and **, p<0.01, indicates a significant difference between the paired groups

black and white squares) then at time ligand t2 stimulated again with HBSS (black squares) or CXCL17 (300 nM, white squares and circles) and change in BRET observed. (b) Change in BRET following application of increasing concentrations of CXCL17 (1 pM -1 µM) 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 (10 nM) in the absence (black bar) or presence of AMD3100 (black squares) or CXCL17 (white circles). (d) HEK293 cells transiently-transfected with CXCR1/Rluc8, CCR5/Rluc8 or β2-adrenoceptor/Rluc8 and β-arrestin2/Venus were stimulated with CXCL17 (300 nM) or either CXCL8 (10 nM), CCL5 (10 nM) or isoprenaline (100 µM) respectively. Corrected BRET was calculated as described in Methods. Points or bars represent mean ± s.e.m. of three (a, b and d) or six (c) individual experiments

Live (a) HEK293 cells or (b) membrane preparations stably-expressing NLuc/CXCR4 were incubated for 1 hr at 37 °C with

white circles). (c) Confocal imaging (Zeiss LSM 710) of HEK293 cells stablyexpressing SNAP/CXCR4 under unstimulated conditions (vehicle) or after treatment with 100 nM CXCL12 or 300 nM CXCL17 (1 h at 37°C). Data are representative of three individual experiments. Scale bar represents 20 μm. (d) Membrane preparations stably-expressing NLuc/CXCR4 were permeabilised with saponin (0.25 mg/ml) and

nM) in the absence (black bar) or presence of AMD3100 (1 µM, grey bar) or CXCL17 (300 nM, hatched bar) white bar (HBSS) is vehicle control. (e) Membrane preparations stably expressing NLuc/CXCR4

pM -10 µM, black squares) or CXCL17 (100 pM -1 µM, white circles). (f) HEK293 cells or (g) membrane preparations stably-expressing NLuc/CXCR4 were incubated for 1 hr at 37 °C with IT1t-BY630/650 (100 nM) and increasing concentrations of AMD3100 (10 pM -10 µM, black squares) or CXCL17 (100 pM -1 µM, white circles)

HEK293 cells expressing genome-edited CXCL12-HiBiT, were transiently transfected with SNAP/CXCR4 were incubated for 1 hr at 37 °C in the absence or presence of increasing concentrations of AMD3100 (black squares, 100 pM -10 µM), CXCL17 (white circles, 100 pM -1 µM) or Surfen (black triangles, 30 nM -30 µM). CXCL12-HiBiT was complemented with purified LgBiT (30 nM) and SNAP/CXCR4 labelled with cell impermeant AlexaFluor488 prior to measurement of BRET. Bars represent basal BRET in the absence of added AlexaFluor488 label