Macrophage GPR35 promotes tumour growth

We had previously reported a reduced incidence of intestinal tumours in Gpr35 ^–/– mice in spontaneous CRC (Apc ^min) and CAC (AOM/DSS) models.10 Selective deletion of Gpr35 in the intestinal epithelium (ie, in Gpr35 ^fl/fl;Vil-Cre or ‘Gpr35 ^ΔIEC’ mice) was sufficient to reduce tumour incidence in both models.10 In both CRC and CAC, individual tumours in Gpr35 ^–/– mice were strikingly smaller compared with Gpr35 ^+/+ tumours (figure 1A). Levels of CXCL-1, VEGF and IL-1β, proangiogenic mediators mainly produced by TAM,28 31 32 were substantially lower in Gpr35 ^–/– compared with Gpr35 ^+/+ tumours (figure 1B). This prompted us to ask whether GPR35 on LysM⁺ cells, which encompasses macrophages and neutrophils,33 might mediate a TME pathway that promotes tumour growth.17 18 We, therefore, generated Apc ^min allele-carrying mice with LysM-specific Gpr35 deletion (Gpr35 ^fl/fl;LysM-Cre;Apc ^min/+, ‘Gpr35 ^ΔMΦ;Apc ^min’) and compared them to their Gpr35-sufficient controls (Gpr35 ^fl/fl;Apc ^min/+, ‘Gpr35 ^fl/fl;Apc ^min’) (figure 1E). Gpr35 ^ΔMΦ;Apc ^min mice developed tumours more than three times smaller than Gpr35 ^fl/fl;Apc ^min mice (figure 1F), although the total number of tumours was not significantly different (figure 1C). Subjecting Gpr35 ^ΔMΦ (ie, without an Apc ^min allele) to AOM/DSS colitis revealed a similarly stark reduction in tumour size compared with their Gpr35 ^fl/fl controls (figure 1F). In this model of CAC, even the total tumour count in Gpr35 ^ΔMΦ mice was half of that in Gpr35 ^fl/fl mice (figure 1D), and associated with a higher survival rate (online supplemental figure 1D). The reduced incidence and size of tumours in Gpr35 ^ΔMΦ mice was not linked to protection from DSS-induced colitis, as weight loss during short-term (ie, 7 days) DSS, nor during the three cycles of AOM/DSS was indifferent between Gpr35 ^+/+ and Gpr35 ^–/–, and Gpr35 ^fl/fl and Gpr35 ^ΔMΦ mice (online supplemental figure 1A–C). Levels of VEGF, CXCL-1 and IL-1β were also markedly lower in inflammatory AOM/DSS tumours of Gpr35 ^ΔMΦ compared with Gpr35 ^fl/fl mice (figure 1G–I). Inflammatory infiltrate was reduced in Gpr35 ^ΔMΦ tumours compared with Gpr35 ^fl/fl tumours (online supplemental figure 1E). Macrophages and neutrophils express Gpr35, which is efficiently deleted in both cell types in Gpr35 ^ΔMΦ mice (online supplemental figure 2B). Of note, GPR35 expression extends to human peripheral blood-derived neutrophils and monocyte-derived macrophages (online supplemental file 2C). Neutrophil activation, measured by tissue MPO levels, were similar in Gpr35 ^ΔMΦ and Gpr35 ^fl/fl mice after 3 cycles of AOM/DSS, suggesting neutrophil GPR35 did not affect neutrophil activation per se in this context (online supplemental figure 1F). Altogether, this demonstrated that GPR35 in LysM⁺ myeloid cells potently promotes tumour growth in both CRC and CAC, and hinted toward an important role of tumour angiogenesis in this microenvironmental pathway.

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Figure 1

GPR35 increases tumour size and release of proangiogenic cytokines. (A) The area of intestinal adenomas from Apc ^min mice either wild-type (Gpr35^+/+ ) and globally deficient for GPR35 (Gpr35^-/- ) (left panel) or from Gpr35^+/+ and Gpr35^-/- mice exposed to AOM and three cycles of DSS (right panel) were compared. N=7 tumours per genotype. (B) CXCL-1, VEGF and IL-1β levels measured in AOM/DSS tumour tissue of Gpr35^+/+ and Gpr35^-/- mice. N=6 tumours per genotype. (C) Mice conditionally deficient for macrophage GPR35 (Gpr35 ^ΔMΦ) were crossed with APC^min mice and tumours were counted when the mice reached an age of 16 weeks. N=14 mice per genotype. (D) Gpr35 ^ΔMΦ mice were exposed to AOM and DSS. Colon adenomas were counted in Gpr35 ^ΔMΦ mice and in their control Gpr35^fl/fl . N=11 to 13 mice per genotype. (E) Representative images showing H&E stain of tumours from Gpr35^fl/fl and Gpr35 ^ΔMΦ mice. (F) The area of intestinal adenomas from either Gpr35^fl/fl ; APC ^min and Gpr35 ^ΔMΦ; Apc ^min mice (left panel) or from Gpr35^fl/fl and Gpr35 ^ΔMΦ mice exposed to AOM/DSS (right panel) were compared. N=9 tumours per genotype. (G–I) CXCL-1 (G), VEGF (H) and IL-1β (I) levels measured in AOM/DSS tumour tissue of Gpr35^fl/fl and Gpr35 ^ΔMΦ mice. N=6 tumours per genotype. All data represented as mean±SEM. Statistical significance was calculated using Mann-Whitney U after Kruskal-Wallis testing. * p<0.05, ** p<0.01. AOM, azoxymethane; APC, Adenomatosis Polyposis Coli; DSS, dextran sodium sulphate; IL-1β, interleukin 1β; n.s., not significant.

GPR35 deletion in LysM⁺ myeloid cells reduces angiogenesis

The architecture of blood vessels in AOM/DSS-induced tumours of Gpr35 ^fl/fl mice appeared to be different from that in tumours of Gpr35 ^ΔMΦ mice. We observed fewer endothelial cells, identified by CD31, which stretched along the adenomatous crypts in Gpr35 ^ΔMΦ mice, whereas in adenomas from Gpr35 ^fl/fl mice, the CD31⁺ neo-vasculature resembled a reticular structure (figure 2A). When these tumours were analysed for total numbers of CD31⁺ cells by FACS, fewer CD31⁺ cells were detected in Gpr35 ^ΔMΦ compared with Gpr35 ^fl/fl tumours (figure 2B). Neither primary CD31⁺ murine endothelial cells, nor human umbilical vein endothelial cells, human microvascular endothelial cells (HMVECs), or the murine endothelial cell lines SVEC40 and H2-11 expressed Gpr35 mRNA (online supplemental figure 2A). We also noticed that adenomas in Gpr35 ^ΔMΦ mice exhibited only sparse staining for CD206, the mannose receptor and a marker of ‘M2’ macrophages, compared with dense staining in Gpr35 ^fl/fl adenomas (figure 2A). FACS analysis of Gpr35 ^ΔMΦ tumours (online supplemental figure 2D) showed significantly less CD68⁺ cells compared with Gpr35 ^fl/fl tumours (figure 2B). The number of CD163⁺ and CD206⁺ cells also trended lower in Gpr35 ^ΔMΦ compared with Gpr35 ^fl/fl tumours (figure 2B). Ly6-G⁺ cell counts were lower in Gpr35 ^ΔMΦ compared with Gpr35 ^fl/fl tumours, consistent with lower immune cell infiltration (online supplemental figure 1E.) Bone-marrow derived Gpr35 ^+/+ macrophages stimulated to differentiate into paradigmatic ‘M0’, ‘M1’ and ‘M2’ phenotypes secreted substantially more of the angiogenic mediators VEGF and CXCL-1 compared with their Gpr35 ^–/– counterparts (figure 2C,D). Human M0, M1 and M2 macrophages, differentiated from induced pluripotent stem cells (iPSC), secreted substantially more VEGF and CXCL-8 when they were differentiated from iPSCs genetically engineered to express the hypermorphic GPR35 T108M risk variant,10 compared with those differentiated from their syngeneic non-risk parent strain (figure 2E,F). This demonstrated that GPR35-dependent angiogenic factor release is conserved across species, and the human risk variant profoundly enhances their secretion. Macrophages involved in repair and (tumour)angiogenesis are thought to resemble mainly an M2 phenotype. We, therefore, pursued endothelial tube formation assays with SVECs or H2-11 murine endothelial cells stimulated with supernatants from Gpr35 ^+/+ and Gpr35 ^–/– M2 macrophages. Conditioned media from Gpr35 ^+/+ M2 macrophages induced SVECs to form significantly longer branches, more junctions within the tubular network, and a total area of formed tubes significantly larger compared with conditioned media from Gpr35 ^–/– macrophages (figure 3A). This suggested that GPR35 controls the release of angiogenic factors in macrophages that promote endothelial tube formation, a prerequisite of neovascularisation. Tube formation only occurs in the presence of growth factors such as VEGF. Control experiments show that supernatants of BMDM contain sufficient amounts of VEGF and potential other soluble factors CXCL-1/CXCL-8. Control RPMI media was not able to induce tube formation in 2 H-11 cells, which recurred on addition of recombinant VEGF (figure 3B). Blocking VEGF with an anti-VEGF antibody reduced branch length, number of junctions and the total mesh area, indicating the importance of growth factors released by macrophages (figure 3C). Increased branching and mesh formation of 2 H-11 endothelial cells (online supplemental figure 3) when exposed to Gpr35 ^+/+ compared to Gpr35 ^–/– macrophage supernatants was associated with increased phosphorylation at Tyr¹¹⁷⁵ of the VEGF receptor (figure 3E and online supplemental figure 5A). HMVEC exposed to supernatants from human T108M M2 macrophages differentiated from iPSCs also exhibited increased branching and mesh formation compared with those from their non-risk counterparts (figure 3D). These data were consistent with GPR35-dependent secretion of factors, including VEGF, from macrophages that act on endothelial cells to promote vessel formation.

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Figure 2

GPR35 deletion in myeloid cells reduces angiogenic potential. (A) Adenomas from Gpr35^fl/fl and Gpr35 ^ΔMΦ mice exposed to AOM/DSS stained for CD31⁺ endothelial cells and CD206⁺ M2 macrophages. N=5 each genotype, representative confocal microscopy. Scale bars 100 µm. (B) FACS analyses of CD31⁺, CD68⁺, CD163⁺, CD206⁺ and Ly-G6⁺ cells in Gpr35^fl/fl and Gpr35 ^ΔMΦ tumour tissue. N=8–12 Gpr35^fl/fl tumours from 8 to 12 mice and N=7 Gpr35 ^ΔMΦ tumours from mice. (C) VEGF levels in M0, M1 and M2 BMDM from Gpr35^+/+ and Gpr35^-/- mice. N=6 per genotype. (D) CXCL-1 levels in M0, M1 and M2 BMDM from Gpr35^+/+ and Gpr35^-/- mice. N=6 per genotype. (E) VEGF levels in supernatants of M0, M1 and M2 human iPS cell-derived macrophages. N=6 each genotype. (F) CXCL-8 levels in supernatants of M0, M1 and M2 human iPS cell-derived macrophages. N=6 each genotype. * p<0.05, ** p<0.01. AOM/DSS, azoxymethane followed by dextran sodium sulphate; iPS, inducible-pluripotent-stem; n.s., not significant; WT, wild type.

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Figure 3

GPR35 deletion in macrophages and neutrophils reduces tube formation. (A) Tube formation assay with murine SVEC endothelial cells incubated with Gpr35^+/+ and Gpr35^-/- M2 macrophage supernatants. Branch length, number of junctions and total mesh area were determined using ImageJ’s angiogenesis tool. N=18. (B) 2 H-11 control tube formation assays with control RPMI/10%FBS and RPMI/10%FBS containing 30 ng/mL VEGF. No macrophage supernatants present. Branch length, number of junctions and total mesh area were determined using the Image J’s angiogenesis tool. N=14 C. 2 H-11 tube formation assays with supernatants of wild-type (WT) and knock-out macrophages in the presence of blocking anti-VEGF antibody. Branch length, number of junctions and total mesh area were determined using Image J’s angiogenesis tool. N=14. (D) HMVEC tube formation assays with human microvascular endothelial cells. Endothelial cells were incubated with supernatants of iPS cell-derived macrophage supernatants (WT and T108M risk variant). Branch length, number of junctions and total mesh area were determined using Image J’s angiogenesis tool. N=10. (E) VEGF receptor 2 phosphorylation of 2 H-11 cells treated with WT or Gpr35^–/– M2 supernatants. All data represented as mean±SEM. Statistical significance was calculated using Mann-Whitney U after Kruskal-Wallis testing. HMVEC, human microvascular endothelial cell; iPS, inducible-pluripotent-stem; n.s., not significant.

GPR35 promotes ex vivo endothelial sprouting

On in vitro culture, fibroblasts and macrophages are the first cells to migrate out of the aortic ring, setting the stage for the formation of neovessels. Endothelial cells typically start migrating and proliferating on day 2–3, and pericytes eventually surround the elongating sprouts.34 35 Here, we tested whether GPR35 is involved in vascular sprouting in vascular tissue and hence subjected aortic rings from wild-type and knock-out mice to an aortic sprout assay. Gpr35 ^–/– aortic rings developed markedly fewer vessel sprouts compared with Gpr35 ^+/+ rings (figure 4A). Addition of exogenous VEGF (30 ng/mL) was required (added on day 1), as only very little sprouting was triggered in wild-type and knock-out aortic rings in its absence (figure 4A). Removal of large parts of the aortic adventitia, which contains macrophages, abrogated the difference between Gpr35 ^+/+ and Gpr35 ^–/– aortic rings and let overall far fewer sprouts develop compared with when the aortic wall was left intact (figure 4B). Vessel sprouting from Gpr35 ^ΔMΦ compared with Gpr35 ^fl/fl aortic rings was equally profoundly suppressed (figure 4C) as in Gpr35^–/– compared with Gpr35 ^+/+ aortic rings. This demonstrated that GPR35 on LysM⁺ myeloid cells orchestrated the outgrowth of neovessels.

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Figure 4

Macrophage GPR35 controls vascular sprouting by interacting with the Na/K-ATPase. (A) Aortic rings from Gpr35 ^+/+ or Gpr35 ^–/– mice embedded in collagen matrix. Number of sprouts counted daily until day 9 of the experiment. Aortic rings were either embedded in media containing 30 ng/mL of VEGF (full circles) or left in OptiMEM media without growth factors (open circles) N=9 mice per genotype. (B) Vascular adventitia was removed from aortic rings from Gpr35 ^+/+ or Gpr35 ^–/–. Number of sprouts on day 9. N=6 mice per genotype. (C) Aortic rings from wildtype animals (Gpr35^fl/fl ) or mice lacking GPR35 conditionally on their LysM⁺ cellsGpr35 ^ΔMΦ. N=13 for Gpr35^fl/fl and 23 for Gpr35^ΔMΦ mice. (D) KYNA (100 µM) was added to the aortic rings of Gpr35 ^+/+ or Gpr35 ^–/– mice and vascular sprouts counted on day 9. N=5 mice for each genotype. (E) CXCL-17 (20 ng/mL) was added to the aortic rings of Gpr35 ^+/+ or Gpr35 ^–/– mice and vascular sprouts counted on day 9. N=6 mice for each genotype. (F) Anti-VEGF antibody (100 ng/mL) was added to the aortic rings of Gpr35 ^+/+ or Gpr35 ^–/– mice and vascular sprouts counted on day 9. n=6 for each genotype. (G) Blocking CXCR2 pepducin x1/2pal-i3 (3 µM) was added to the aortic rings of Gpr35 ^+/+ or Gpr35 ^–/– mice and vascular sprouts counted on day 9. n=6 for each genotype. (H, I.) ouabain (100 µM) was added to the aortic rings of Gpr35 ^+/+ or Gpr35 ^–/– mice (H) and to the aortic rings of Gpr35^fl/fl and Gpr35 ^ΔMΦ mice (I) and vascular sprouts counted on day 9. N=6 for each genotype. (J, K) pNaKtide (1 µM) was added to the aortic rings of Gpr35 ^+/+ or Gpr35 ^–/– mice (J) and to the aortic rings of Gpr35^fl/fl and Gpr35 ^ΔMΦ mice (K) and vascular sprouts counted on day 9. N=9 for each genotype. All data represented as mean±SEM. Statistical significance was calculated using Mann-Whitney U after Kruskal-Wallis testing.* p<0.05, ** p<0.01. n.s., not significant.

KYNA, a product of tryptophan catabolism, and the chemokine CXCL-17 have been suggested as ligands of GPR35.36 37 Recombinant CXCL-17, added to aortic ring cultures, increased vessel sprouting in both Gpr35 ^+/+ and Gpr35 ^–/– aortic rings, indicating that CXCL-17 signals in a GPR35-independent manner (figure 4E). KYNA did not alter vessel sprouting, suggesting it is not involved in GPR35-mediated angiogenesis (figure 4D). Addition of a neutralising anti-VEGF monoclonal antibody on day 2 of the experiment, that is, after initial exogenous triggering, reduced vessel sprouting in Gpr35 ^+/+ aortic rings to levels observed in Gpr35 ^–/– aortic rings, in which sprouting was not affected (figure 4F). Moreover, blocking CXCL-1 signalling by blocking its receptor CXCR2 with a selective pepducin38 also reduced vessel sprouting from Gpr35 ^+/+, but not from Gpr35 ^–/– aortic rings (figure 3G). This demonstrated that GPR35 promotes vessel sprouting via VEGF and CXCR2, which tallied with higher VEGF and CXCL-1 secretion in tissue and macrophages from Gpr35 ^+/+ compared to Gpr35 ^–/– mice.

GPR35 promotes angiogenesis via the sodium-potassium-pump in macrophages

Reduced Na/K-ATPase ion pumping activity in the absence GPR35 elevates intracellular Ca²⁺ and lowers K⁺ levels.10 The endogenous cardiotonic steroid ouabain achieves a similar effect via binding to Na/K-ATPase and retaining it in an inactive (E2P) state.39 Ouabain indeed reduced vessel sprouting in Gpr35 ^+/+ aortas to levels observed in Gpr35 ^–/– aortas, whereas sprouting was not significantly changed but slightly increased in Gpr35 ^–/– aortas (figure 4H). This was precisely phenocopied in aortas from Gpr35 ^fl/fl when compared with Gpr35 ^ΔMΦ mice (figure 4I), demonstrating vessel sprouting was coordinated by GPR35 on myeloid cells in a Na/K-ATPase-dependent manner. Among its non-pump functions, Na/K-ATPase acts as a scaffold for Src kinase and is thereby important in signal transduction.11 40 41 The α1 type Na/K-ATPase forms a complex with the Src SH2 and kinase domain and keeps the kinase domain in an inactive state.11 A 20-amino acid sequence (pNaKtide) specifically inhibits Na/K-ATPase-associated Src activation.42 43 Addition of pNaKtide peptide decreased sprouting in Gpr35 ^+/+ and Gpr35 ^fl/fl aortas to levels observed in their respective Gpr35 ^–/– and Gpr35 ^ΔMΦ counterparts, in which pNaKtide had no further effect (figure 4J,K). When exposing Gpr35 ^+/+ macrophages to ouabain, no increase in Src phosphorylation was observed. This was unexpected given previously published work in cell lines that do not endogenously express GPR35.43 Src phosphorylation was indeed increased by ouabain in Gpr35 ^–/– macrophages (online supplemental figure 4), pointing towards an important role of GPR35 determining the Src signalling response on pharmacological inhibition of Na/K-ATPase. Altogether this demonstrated that GPR35 expressed on myeloid cells within the aortic wall promotes vessel sprouting in a manner dependent on Na/K-ATPase-triggered Src activation.

GPR35-deficient tumours produce less MMPs

A link between MMP-9-positive TAMs and endothelial cells responding to angiogenic induction has been established.44 To assess MMP activity, we performed gelatine zymography of extracts from tumours arising in the AOM/DSS model. Gpr35 ^–/– tumours exhibited much lower gelatinolytic activity compared with Gpr35 ^+/+ tumours (figure 5A). Addition of 4-aminophenylmercuric acetate (APMA), which activates pro-MMPs to active zymogens, to tumour extracts did not further increase their gelatinolytic activity (figure 5A). This indicated that essentially all available MMP activity in tumours is already in the active form. Macrophages can release MMPs and tissue inhibitors of MMPs. While native supernatants from Gpr35 ^–/– or Gpr35 ^+/+ M1 and M2 BMDMs did not exhibit MMP activity (data not shown), addition of APMA to supernatants revealed substantial activity in Gpr35 ^+/+ and much lower activity in Gpr35 ^–/– macrophage supernatants (figure 5B). Ouabain and pNaKtide treatment of Gpr35 ^+/+ M2 macrophages reduced their supernatants’ MMP activity (figure 5C), whereas it did not affect MMP activity in Gpr35 ^–/– supernatants (figure 5C). Differences in zymography were detected in gelatine- but not casein gels. We, therefore, measured the gelatinases MMP2 and MMP9 in tumour tissue and supernatants from BMDM. Gpr35 ^ΔMΦ tumours contained substantially less MMP2 and MMP9 (figure 5D) compared with AOM/DSS-induced tumours from Gpr35^fl/fl mice. M2 macrophages from Gpr35 ^–/– mice correspondingly contained less MMP2 and MMP9 compared with their Gpr35 ^+/+ counterparts (figure 5E). Moreover, supernatants from human iPSC-derived M0 and M2 macrophages carrying the T108M risk variant contained higher amounts of MMP2 compared with those from non-risk variant cells, with MMP2 levels in T108M variant M1 macrophages levels also trending higher (figure 5F). Altogether, this implied that GPR35 controlled via Na/K-ATPase-dependent Src activation the level of MMP activity in tumours, with the T108M variant yielding highest levels.

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Figure 5

Lack of GPR35 results in reduced MMP levels. (A) MMP levels in Gpr35 ^+/+ and Gpr35 ^–/– adenomas. Tissue extract MMPs were activated with APMA. Area under the curve was analysed using image J. N=6 tumours per genotype. (B) MMP levels in M1 and M2 BMDM supernatants from Gpr35 ^+/+ and Gpr35 ^–/– mice. Supernatants were APMA-activated. area under the curve was analysed using image J N=9 mice per genotype. (C) MMP levels in M2 macrophages pretreated with ouabain (100 µM) or pNaKtide (1 µM). Area under the curve was analysed using image J N=6 mice per genotype. (D) Total (inactive zymogen and active enzyme) MMP2 and MMP9 levels in tumour tissue from Gpr35^fl/fl and Gpr35 ^ΔMΦ mice. (E) MMP2 and MMP9 levels from M2 murine BMDM supernatants. (F) Total (inactive zymogen and active enzyme) MMP2 levels from supernatants of human iPS cell-derived macrophages. Macrophages were polarised to M0, M1 and M2 macrophages. All data represented as mean±SEM. Statistical significance was calculated using Mann-Whitney U after Kruskal-Wallis testing. * p<0.05, ** p<0.01/ APMA, aminophenylmercuric acetate; MMP, matrix-metallo-proteinase; n.s., not significant; WT, wild type.

GPR35 increases endothelial transmigration of monocytes

Circulating CCR2⁺ inflammatory monocytes enter the TME, proliferate and differentiate into TAMs,45 a process that requires endothelial adhesion and transmigration.46 We therefore investigated transmigration by studying myeloid cells harvested from 5-day bone marrow cultures, at which point cells exhibit a monocyte phenotype.47 Mimicking monocyte transmigration, we plated endothelial cells onto Transwell membranes and used supernatants from Gpr35 ^+/+ or Gpr35 ^–/– M2 macrophages as chemoattractant. Gpr35 ^+/+ or Gpr35 ^–/– monocytes added to the upper compartment of the chamber migrated through the endothelial cell layer towards the macrophage supernatants. Compared with Gpr35 ^+/+ monocytes, transmigration of Gpr35 ^–/– monocytes was reduced (figure 6A, left panel). Fewer Gpr35 ^–/– than Gpr35 ^+/+ monocytes adhered to monolayers of H2-11 murine endothelial cells (figure 6A, right panel). Hence GPR35 deficiency in monocytes impaired their ability to adhere to, and transmigrate through, endothelial cells. In converse experiments, we exposed endothelial cells to supernatants of either Gpr35 ^+/+ or Gpr35 ^–/– M2 macrophages, and monocytes of either genotype migrated towards wild-type macrophage conditioned media. Monocytes adhered less to endothelial cells that were exposed to Gpr35 ^–/– M2 macrophage supernatant than to those exposed to Gpr35 ^+/+ supernatant (figure 6B right panel). Transmigration of monocytes through an endothelial monolayer incubated with Gpr35 ^–/– M2 macrophage conditioned media was correspondingly reduced when compared with migration through endothelial cells exposed Gpr35 ^+/+ supernatants. Fewer Gpr35 ^–/– than Gpr35 ^+/+ monocytes migrated through monolayers treated with either Gpr35 ^–/– or Gpr35 ^+/+ macrophage supernatants (figure 6B, left panel). This demonstrated that GPR35 on macrophages controls the release of factors that promoted adhesion and transendothelial migration of monocytes.

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Figure 6

Lack of GPR35 decreases endothelial transmigration. (A) Right panel: Gpr35 ^+/+ and Gpr35 ^–/– monocytes transendothelial migration towards conditioned media from Gpr35 ^+/+ and Gpr35 ^–/– M2 macrophages in the lower compartments of migration chambers. Untreated endothelial cell monolayer. Left panel: adhesion of Gpr35 ^+/+ and Gpr35 ^–/– monocytes to 2 H-11 murine endothelial cells. Untreated endothelial cell monolayer. N=6 mice per genotype. (B) Left panel: transendothelial migration of monocytes through monolayers of 2 H-11 murine endothelial cells exposed to conditioned media from Gpr35 ^+/+ and Gpr35 ^–/– M2 macrophages. right panel: adhesion of Gpr35 ^+/+ and Gpr35 ^–/– monocytes to 2 H-11 murine endothelial cells treated with conditioned media from Gpr35 ^+/+ and Gpr35 ^–/– M2 macrophages. N=6 mice per genotype. (C) Upper panel: VCAM-1 expression in 2 H-11 cells treated with conditioned media from Gpr35 ^+/+ and Gpr35 ^–/– M2 macrophages pretreated with control RPMI, ouabain (100 µM) or pNaKtide (1 µM). Lower panel: VCAM-1 expression in Gpr35^fl/fl and Gpr35 ^ΔMΦ AOM/DSS tumours. (D) ERK1/2 phosphorylation of 2 H-11 cells treated with conditioned media from Gpr35 ^+/+ and Gpr35 ^–/– M2 macrophages pretreated with control RPMI, ouabain (100 µM) or pNaKtide (1 µM). (E) Src and ERK1/2 phosphorylation in Gpr35 ^+/+ and Gpr35 ^–/– AOM/DSS tumours. N=3 tumours per genotype. (F) Numbers of macroscopic and microscopic tumours of either g35i2 or vehicle treated mice with AOM/DSS induced tumours. N=8–9 mice/group. (G) AOM/DSS tumour tissue from g35i2 or vehicle treated mice probed for Src and ERK1/2 phosphorylation. N=3 tumours per genotype. (H) CXCL-1, VEGF, MMP2, MMP9 and inflammatory infiltrate in tumour tissue from g35i2 or vehicle-treated wild-type mice. N=6 per genotype for CXCL-1, VEGF, MMP2 and MMP9 ELISA assays, and n=8 for histological assessment of inflammatory cell infiltrate. All data represented as mean±SEM. Statistical significance was calculated using Mann-Whitney U after Kruskal-Wallis testing. AOM/DSS, azoxymethane followed by dextran sodium sulphate; n.s., not significant.

VCAM-1 is a major endothelial cell adhesion molecule that facilitates monocyte transmigration from the peripheral blood into the surrounding tissue.48 VCAM-1 expression was higher in H2-11 endothelial cells exposed to Gpr35 ^+/+ compared with Gpr35 ^–/– M2 macrophage supernatants (figure 6C). This increase was abrogated when M2 macrophages were treated overnight with ouabain or pNaKtide and then washed, prior to collecting supernatants over the ensuing 24 hours (figure 6C and online supplemental figure 5B). In contrast, ouabain or pNaKtide treatment of Gpr35 ^–/– M2 macrophages did not affect endothelial VCAM-1 expression. This demonstrated that increased VCAM-1 induction on endothelial cells was dependent on GPR35, Na/K-ATPase and Src activity in macrophages. Consistent with such GRP35-dependent regulation, VCAM-1 expression in tissue was higher in Gpr35^fl/fl compared with Gpr35 ^ΔMΦ mice (figure 6C). Activation of extracellular signal-regulated kinases (ERK) 1 and 2 by VCAM-1 is critical for endothelial activation, transmigration and angiogenesis.48 Conditioned media from Gpr35 ^–/– M2 macrophages induced less ERK1/2 phosphorylation in H2-11 endothelial cells compared with Gpr35 ^+/+ supernatants (figure 6D and online supplemental figure 5C). Similar to VCAM-1 expression, elevated ERK1/2 phosphorylation in H2-11 endothelial cells exposed to Gpr35 ^+/+ macrophages was prevented by pretreatment of macrophages with ouabain or pNaKtide (figure 6D). In contrast, endothelial ERK1/2 phosphorylation was not affected when triggered by supernatants of inhibitor-treated Gpr35 ^–/– macrophages (figure 6D). Src and ERK1/2 phosphorylation was correspondingly lower in Gpr35 ^–/– compared with Gpr35 ^+/+ tumours (figure 6E and online supplemental figure 5D). This hence demonstrated that GPR35-modulated activation of Na/K-ATPase, and Na/K-ATPase-dependent Src activation, in macrophages controlled Erk1/2 phosphorylation and VCAM-1 expression on endothelial cells via transmissible signals.

Gpr35 blockade with a GPR35 specific pepducin reduces tumour size in the AOM/DSS-induced CAC model

We have previously shown that treatment of wild-type mice with a GPR35-selective, inhibitory pepducin (g35i2) reduced the number of inflammatory colon tumours.10 In the AOM/DSS model, the number of macroscopically visible tumours in g35i2-treated wild-type mice decreased fivefold, whereas the number of microscopic tumours decreased only threefold (figure 6F). Src Tyr⁴¹⁶ phosphorylation in AOM/DSS tumours was substantially lower in g35i2- compared with control-treated wild-type mice, and was accompanied by reduced Thr²⁰²/Thr²⁰⁴ phosphorylation of ERK1/2 (figure 6G). Furthermore, CXCL-1, VEGF, MMP2 and MMP9 levels as well as immune cell infiltration were significantly decreased when wildtype mice were treated with the g35i2 compound (figure 6H). Altogether this demonstrated that pharmacological GPR35 blockade reduced tumour size, involving the Na/K-ATPase—Src pathway delineated herein.