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Atlas of the anatomical localization of atypical chemokine receptors in healthy mice [1]

['Serena Melgrati', 'Institute For Research In Biomedicine', 'Università Della Svizzera Italiana', 'Bellinzona', 'Graduate School For Cellular', 'Biomedical Sciences', 'University Of Bern', 'Bern', 'Egle Radice', 'Rafet Ameti']

Date: 2023-05

Atypical chemokine receptors (ACKRs) scavenge chemokines and can contribute to gradient formation by binding, internalizing, and delivering chemokines for lysosomal degradation. ACKRs do not couple to G-proteins and fail to induce typical signaling induced by chemokine receptors. ACKR3, which binds and scavenges CXCL12 and CXCL11, is known to be expressed in vascular endothelium, where it has immediate access to circulating chemokines. ACKR4, which binds and scavenges CCL19, CCL20, CCL21, CCL22, and CCL25, has also been detected in lymphatic and blood vessels of secondary lymphoid organs, where it clears chemokines to facilitate cell migration. Recently, GPR182, a novel ACKR-like scavenger receptor, has been identified and partially deorphanized. Multiple studies point towards the potential coexpression of these 3 ACKRs, which all interact with homeostatic chemokines, in defined cellular microenvironments of several organs. However, an extensive map of ACKR3, ACKR4, and GPR182 expression in mice has been missing. In order to reliably detect ACKR expression and coexpression, in the absence of specific anti-ACKR antibodies, we generated fluorescent reporter mice, ACKR3 GFP/+ , ACKR4 GFP/+ , GPR182 mCherry/+ , and engineered fluorescently labeled ACKR-selective chimeric chemokines for in vivo uptake. Our study on young healthy mice revealed unique and common expression patterns of ACKRs in primary and secondary lymphoid organs, small intestine, colon, liver, and kidney. Furthermore, using chimeric chemokines, we were able to detect distinct zonal expression and activity of ACKR4 and GPR182 in the liver, which suggests their cooperative relationship. This study provides a broad comparative view and a solid stepping stone for future functional explorations of ACKRs based on the microanatomical localization and distinct and cooperative roles of these powerful chemokine scavengers.

Funding: The study was supported by grants from Swiss National Science Foundation ( https://www.snf.ch Sinergia CRSII3_160719 ((MT, AR) and 310030_182727 (MT)), the Novartis Foundation for medical-biological Research ( https://www.novartisfoundation.org/ #17B098 (MT)). Fondazione Fidinam, Lugano ( https://www.fidinam.com/it/fondazione-fidinam MT) and the Helmut Horten Foundation ( https://www.helmut-horten-stiftung.org/ MT) also provided support. Wellcome Trust Investigator Award 200817/Z/16/Z (A.R.) and Versus Arthritis Endowment (AR). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2023 Melgrati et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Previous studies have shown that ACKR3, ACKR4, and GPR182 potentially share expression in endothelial cells. However, a wide-ranging map of active expression on structures where these receptors could truly act as scavengers have been missing, in large part due to the absence of specific validated functional antibodies. Through the use of double ACKR-expression reporter mice, ACKR-specific chimeric chemokines and a novel specific ligand for GPR182, we have generated an atlas of expression and coexpression of ACKR3, ACKR4, and GPR182 in healthy young mice. We focused primarily on endothelial cells, where these ACKRs can bind, internalize, and degrade readily available circulating chemokines to maintain tissue homeostasis. Noteworthy, these receptors share the ability to scavenge homeostatic chemokines.

Recently, a novel potential ACKR was identified. GPR182 is closely related to ACKR3 by phylogeny and was initially thought to be the receptor for adrenomedullin, a notion that has subsequently been refuted [ 49 , 50 ]. Two studies have demonstrated both in vitro and in vivo that GPR182 is able to bind and scavenge the chemokines CXCL9, CXCL10, CXCL12, and CXCL13 and could potentially interact with others [ 51 , 52 ]. GPR182 has been found to be preferentially expressed in vascular ECs, liver sinusoidal ECs, LECs, and intestinal stem cells and is up-regulated in tumor-associated LECs [ 53 – 55 ]. It has been found to act as an ACKR to prevent hematopoietic stem cell egress from the BM [ 51 ], and as a chemokine scavenger in the tumor microenvironment to limit T cell infiltration [ 52 ], and also as a negative regulator of hematopoiesis in zebrafish and mice, by regulating the leukotriene B4 biosynthetic pathway [ 56 ]. The recent discovery of GPR182 function as a chemokine scavenger that appears not to signal through G-proteins (51;52) should warrant it being renamed ACKR5.

There are no selective chemokines for ACKRs as they share their ligands with one or more conventional chemokine receptor. To demonstrate ACKR activity in the presence of the respective conventional receptors and to overcome problems with the paucity of specific antibodies, we engineered fluorescent chimeric chemokines, namely CXCL11_12, which selectively binds ACKR3 [ 48 ], and CCL25_19, which selectively binds ACKR4 [ 36 ]. The chimeras are composed of the N-terminus of CXCL11 or CCL25 and the body of CXCL12 or CCL19, respectively, and contain at the C-terminus a tag for site-specific enzymatic labelling. The chimeras were used to demonstrate scavenging by the ACKRs in vivo.

Finally, ACKR4, previously known as CCRL1, is able to bind and scavenge the chemokines CCL19, CCL20, CCL21, CCL22, and CCL25 [ 35 – 37 ]. It has been detected in cortical thymic epithelium, in keratinocytes, in the lung, and in lymphatic endothelial cells (LECs) [ 38 – 40 ]. Expression on the ceiling of the subcapsular sinus (SCS) LECs (cLECs) has been demonstrated to be essential for shaping CCL21 gradients and, consequently, lymph node (LN) homing of dendritic cell as well as the reentry of B cells from the SCS back into the germinal centers [ 41 – 43 ]. ACKR4 has also been found to be critical for T cell migration in inflamed afferent lymphatics [ 44 ]. It was recently identified in mouse spleens in a three-dimensional sinusoidal network connected to the marginal sinus (MS), named the “peri-marginal sinus,” which tightly surrounds the MZ [ 45 ]. ACKR4 mRNA expression was reported in germinal center B cells (GCB) and genetic ablation of the receptor in mice was associated with a hyperactivated B cell phenotype in ACKR −/− plasma blasts and GCBs [ 39 , 46 ], a finding that was not observed with another ACKR4-deficicent mouse strain (ACKR GFP/GFP ) [ 38 , 47 ].

ACKR3, previously named CXCR7, is mainly responsible for scavenging CXCL12 and CXCL11, resulting in their degradation [ 22 – 24 ]. ACKR3 expression was reported in a variety of cells and tissues including neurons, hematopoietic cells, platelets, umbilical vein endothelium and vascular endothelium, T cells, memory B cells, plasma blasts, and marginal zone (MZ) B cells [ 23 , 25 – 29 ]. The importance of ACKR3 is mainly attributed to its ability to scavenge CXCL12, and indeed, ACKR3 blockade increases CXCL12 levels systemically [ 30 , 31 ]. This scavenger receptor is necessary for survival, as global knockout (KO) in mice is perinatal lethal, due to a stenotic cardiac valve phenotype [ 32 ]. Moreover, it was demonstrated to be required for proper migration of primordial germ cells in zebrafish [ 33 , 34 ]. Tight regulation of CXCL12 availability is critical to maintain homeostasis in leukocyte and stem/progenitor cell retention and egress from the bone marrow (BM) and recruitment to sites of inflammation. Vascular endothelial expression of ACKR3 has been reported, allowing its immediate access to circulating CXCL12 in the bloodstream and suggesting that it is indeed acting as a scavenger for CXCL12 [ 31 ].

ACKRs contribute to cell migration by binding and internalizing chemokines, delivering them to lysosomal degradation. This orchestrates the formation of locally confined gradients, which are necessary for efficient cell trafficking [ 3 ]. ACKRs are seven-transmembrane domain molecules that are structurally and phylogenetically related to G-protein–coupled receptors (GPCRs) but upon ligand binding are unable to signal though G-proteins, unlike conventional chemokine receptors [ 4 ]. Given the lack of G-protein activation, ACKR3 was proposed to have a signaling bias towards β-arrestins [ 5 ]. However, recent studies have challenged this hypothesis showing that β-arrestins are dispensable for ACKR3 internalization [ 6 , 7 , 8 – 10 ]. Four human and mouse ACKRs are currently known: ACKR1 through ACKR4. ACKR1, formerly known as Duffy Antigen Receptor for Chemokines (DARC), binds several inflammatory CC and CXC chemokines [ 2 , 11 ]. It is the most different and phylogenetically distant to the other ACKRs and canonical chemokine receptors [ 12 ]. ACKR1 expression was observed notably in erythrocytes, where it acts as a chemokine sink and as a reservoir and in nucleated erythroid cells where it regulates hematopoiesis [ 13 ]. In mouse venular endothelium, ACKR1 internalizes chemokines from the basolateral side and through transcytosis presents them at the luminal side [ 14 ]. ACKR2, also known as D6 or CKBP2, is also able to bind numerous inflammatory chemokines of the CC family [ 2 , 15 ]. More recently it was shown that ACKR2 can also bind the CXC chemokine CXCL10 [ 16 ]. Unlike ACKR1, ACKR2 is a scavenger receptor, which internalizes chemokines and delivers them for lysosomal degradation [ 17 ]. ACKR2 expression has been previously reported in the lung, skin, placenta syncytiotrophoblasts, lymphatic endothelium, and innate-like B cells [ 18 – 21 ].

During inflammation and homeostasis, leukocyte trafficking is chiefly orchestrated by the chemokine system, along with their cognate and atypical receptors [ 1 ]. Chemokines are small (8 to 10 kDa) chemotactic cytokines that guide migrating cells during homeostasis and inflammation. Chemokines are classified into 4 families, based on the position of the first 2 of 4 conserved cysteines, which form disulfide bonds: CC, CXC, CX 3 C, and XC [ 2 ]. Chemokines can promiscuously bind multiple canonical receptors, which trigger cell migration, or atypical chemokine receptors (ACKRs), which mainly act as scavengers.

BM was flushed from long bones with 5-ml RPMI containing 10% FBS. Single-cell suspensions were strained using a 70-μm cell strainer (Corning, Cat: CLS431751), centrifuged, and resuspended in ACK Lysing buffer according to the manufacturer’s instructions (Gibco, Cat: A1049201) for red blood cell lysis. Cells were then stained with antibodies. For intracellular staining, cells were fixed and permeabilized using Cytofix/Cytoperm solution (BD Biosciences, Cat: 554722), then stained with antibodies. Cells were analyzed by flow cytometry (Fortessa, BD).

Freshly harvested LNs were isolated from mice and incubated in digestion mix containing RPMI, Liberase TL (Roche, Cat: 5401020001), DNAse I (Sigma-Aldrich, Cat: 10104159001) at 37°C for 1 hour with occasional shaking and thorough mixing. Single-cell suspensions were strained using a 70-μm cell strainer (Corning, Cat: CLS431751), centrifuged, and resuspended in αMEM (no nucleosides, + L-glutamine, Gibco, Cat: 12561056) supplemented with 10% FBS and 1% Penicillin/Streptomycin. Cells were then stained with antibodies and analyzed by flow cytometry (Fortessa, BD).

RNA was extracted from sorted CD45 − CD31 + GPR182-mCherry + or GPR182-mCherry − or ACKR3-GFP pos , ACKR3-GFP mid , or ACKR3-GFP neg spleen cells using Quick-RNA Microprep kit (Zymo Research, Cat. R1050), following manufacturer’s instructions. Briefly, pellets were lysed in RNA lysis buffer and purified in Zymo-Spin IC columns. DNAse was added to remove DNA contaminants. cDNA was reverse transcripted using qScript cDNA SuperMix (QuantaBio, Cat: 95048–025). RT-qPCR was performed using PerfeCTa SYBR Green FastMix (QuantaBio, cat: 95072–012), and ΔΔCT calculated using GAPDH as housekeeping gene.

Freshly harvested spleens were injected with a digestion cocktail containing 0.1 mg/ml Dispase Grade I (Roche, Cat: D4818), 0.2 mg/ml Collagenase IV (Roche, Cat: 11088866001), 0.025 mg/ml DNAse I (Sigma-Aldrich, Cat: 10104159001) in RPMI 1640 (Gibco), and incubated at 37°C for 30 minutes. Spleens were transferred into fresh digestion mix and minced into small pieces, then incubated at 37°C for 20 minutes. The suspension was thoroughly mixed every 7 to 10 minutes. Digestion was quenched by adding cold Quench buffer (5 mM EDTA, 3% FBS in PBS). Cells were washed, and red blood cells lysed in ammonium-chloride-potassium lysis buffer. CD45 + cells were removed using CD45 MicroBeads (Miltenyi Biotec, Cat: 130-052-301) and LS columns (Miltenyi Biotec, Cat: 130-042-401) following manufacturer’s instructions. Cells were then stained with antibodies and analyzed by flow cytometry (Fortessa, BD).

Mice were injected IV through the tail vein with a 150-μl solution containing 2.5-μM fluorescent chemokine(s) in PBS. Animals were killed in CO 2 30 minutes later, perfused, and organs harvested as described above.

For BM slice preparation, femurs and tibiae were isolated, cleaned, and immersed in 2% PFA for 6 hours at 4°C, then dehydrated in 30% sucrose for 72 hours at 4°C, as previously described [ 62 ]. Bones were embedded in OCT and snap frozen in liquid nitrogen. Bones were sectioned longitudinally using a cryostat until the BM cavity was exposed. The OCT block containing the sample was reversed to repeat the procedure on the opposite side until the cavity was visible. Samples were incubated in blocking solution containing 0.2% Triton X-100, 1% fatty acid–free bovine serum albumin (BSA), 10% rabbit (Gibco, 16120–107) or goat serum (Gibco, 16210–064) in PBS overnight at 4°C. Primary antibody staining were performed in blocking solutions for 3 days at 4°C. Samples were then washed in PBS and incubated in blocking solution containing secondary antibodies for 3 days. Following washing steps in PBS, samples were then cleared in RapidClear for 6 hours. Samples or sections were imaged using either a Leica SP5 or a Stellaris 8 confocal microscope.

For tissue clearing, agarose-embedded organs were sectioned using a Leica Vibratome to produce 500-μm slices. Sections were permeabilized and blocked in PBS containing 2% Tx-100, 10% serum, 0.05% NaN 3 ) for 24 hours at RT, then stained with antibodies over 48 hours at 4°C. Sections were washed in wash buffer (PBS, 1% FBS, 0.1% Tx-100, 0.01% NaN 3 ) for 1 hour 3 times at RT. Sections were then cleared in RapiClear 1.52 (SUNJin Lab, Cat: #RC152001) until transparent following manufacturer’s instructions. Alternatively, fixed spleens were washed in PBS and incubated overnight at 4°C in a hydrogel solution (4% acrylamide and 0.25% of 2,2′-Azobis[2-(2-imidazolin-2-yl)-propane]-dihydrochloride in PBS). The solution containing the spleens was saturated with nitrogen to remove oxygen and incubated at 37°C for polymerization. Spleens were washed in PBS and cleared with the ACT-ECT method [ 61 ] (active clarity technique–electrophoretic tissue clearing containing 4% SDS and 200 mM boric acid (Logos)) for 5 hours. Spleens were washed overnight with PBS and incubated for 2 hours with cubic mounting media (50% sucrose, 25% urea, and 25% N,N,N′,N′-tetrakis-(2-hydroxypropyl)-ethylenediamine) to increase transparency.

Cross-sections of colon and small intestines were removed, cleaned, and fixed in 2% PFA/PBS for 4 hours followed by 10%, 15%, and 30% sucrose/PBS for 3 hours or overnight. Tissues were subsequently frozen in optimum cutting temperature (OCT) compound on dry ice and stored at −80°C. Around 5-μm thin sections were cut on a cryomicrotome, air dried, and then stored at −20°C. Before staining, sections were rehydrated in 0.1% BSA/PBS and blocked with 10% goat serum for 30 minutes. Antibodies were diluted in 0.1% BSA/PBS and incubated on the sections for 40 minutes at RT. After 3 washes in 0.1% BSA/PBS, the sections were stained with DAPI (MilliporeSigma) for 5 minutes. After another washing step, the sections were mounted with Prolong Gold (Thermo Fisher Scientific) and cured overnight. Sections were imaged on a LSM800 (Zeiss).

Mice were killed in CO 2 and perfused first with 30 ml PBS, then with 10 ml PBS containing 2% PFA (MerckMillipore). Organs were harvested and fixed for 4 to 16 hours in 4% PFA at 4°C on a shaking platform. Organs were washed in PBS and embedded in 3% low-gelling temperature agarose (Sigma, Cat: A9414). Tissues were sectioned using a Leica Vibratome to produce 50 to 150 μm slices. Sections were blocked in Blocking Buffer (PBS, 1% FBS, 0.1% Tx-100, 0.01% NaN 3 ) for 1 hour at RT and then stained with selected primary or directly conjugated antibodies overnight in Blocking Buffer. Following two 20-minute washes, sections were stained using secondary antibodies (1 hour RT) or DAPI (15 minutes RT), washed twice, and mounted on glass slides in FluoroMount (Sigma, Cat: F4680).

All animals were housed in specific pathogen-free facility, and all experiments were performed in accordance with the Swiss Federal Veterinary Office guidelines and authorized by the Animal Studies Committee of Cantonal Veterinary (Cantonal Committee for Animal Experimentation (CCEA, License: TI-33/2020), and under the license number PA672E0EE issued to QMUL by the UK Home Office. In addition, ACKR4 GFP/+ reporter mice were housed in specific pathogen-free facilities at Queen Mary University London, Charterhouse Square, or University of Birmingham, Edgbaston, UK. Experiments were performed using the tissues of 8- to 12-week-old mice, as approved by the Institutional Ethics and Animal Welfare Committees and the Home Office, UK. All mice used were in the C57BL/6 background. For the experiments, animals of both genders were used with no sex-dependent differences observed.

GPR182 mCherry/+ mice were generated by CRISPR/Cas9 genome engineering in C57BL/6J mouse embryos, with mCherry knock-in replacing 1 GPR182 allele, resulting in a heterozygous reporter animal ( S1 Methods , S1 Fig ) [ 59 , 60 ]. By crossing 2 heterozygous reporter mice, we obtained GPR182-KO offspring (in mendelian ratio), in which both alleles of GPR182 were replaced with mCherry in homozygosity. Animals were crossed with ACKR3 GFP/+ and ACKR4 GFP/+ to obtain double reporter mice.

For binding and uptake assays, 300.19 pre-B cells stably transfected with receptors (and GFP in a T2A system), or parental, were incubated with 300 nM chimeric chemokines (all labeled in AF647 or Dy649) for 45 minutes. Cells were washed and analyzed by FACS (Fortessa, BD). The MFI of GFP, which is proportional to the level of receptor expressed [ 58 ], was used to normalize.

Mouse 300.19 pre-B cells were cultured in B cell medium containing RPMI-1640 supplemented with 10% FBS, 1% PenStrep, 1% nonessential amino acids, 1% Glutamax, and 50 μM β-mercapto ethanol (β-ME). All cell culture media and supplements were from Gibco/Thermo Fisher. Cells stably expressing ACKRs or GPR182 were transfected using Amaxa Nucleofector (Lonza), along with a T2A-GFP sequence. Receptors were expressed fused via a self-cleaving peptide to green fluorescent protein (GFP), which splits posttranslationally to produce the 2 proteins and mark cells expressing the receptor.

Results

Expression in intestine and colon Analysis of intestine and colon revealed common and distinct patterns of ACKR expression (Figs 6 and S5). In the small intestine, GPR182 was mainly expressed in the villi in lacteals (Lyve-1+) and in the lymphatic vessels of the submucosa, and only marginally in CD31+ capillaries in the villi (Figs 6A, 6B, S5A and S5B). ACKR3, instead, was found in CD31+ endothelium of both the villi and the submucosa of the small intestine (Fig 6A). In contrast, ACKR4 was found in non-endothelial Lyve-1 and CD31-negative cells of the submucosa (Fig 6B). Closer inspection revealed that ACKR4+ cells were present above, below, and within the outer longitudinal smooth muscle cell layer, as well as within the serosal layer (Fig 6C), in direct contact with interstitial c-kit+ Cajal cells (Fig 6D) and colocalized with vimentin, suggesting their mesenchymal origin (S5E Fig). In the colon, ACKR3 was primarily found in the colonic vasculature (Figs 6E and S5C). Some coexpression with GPR182 was seen; however, the latter was mainly found in the submucosa, while ACKR3 was observed mostly in the vessels of the mucosa of the colon. Conversely, ACKR4 expression in the colon resembled its unique expression in the small intestine, namely in the submucosa (above, below, and within) and subserosa (Figs 6F, 6G and S5D). As in the small intestine, these cells were found in close proximity to Cajal cells and were in part vimentin positive (Figs 6H and S5E). PPT PowerPoint slide

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TIFF original image Download: Fig 6. Expression of chemokine scavengers in small intestine and colon. Confocal imaging of small intestine from (A) ACKR3GFP/+ − GPR182mCherry/+ mouse, GPR182 (mCherry, red), ACKR3 (GFP, green), CD31 (pink), and Lyve-1 (cyan) (scale bar = 70 μm), and (B) ACKR4GFP/+ − GPR182mCherry/+ mouse, GPR182 (mCherry, red), ACKR4 (GFP, green), CD31 (pink), and Lyve-1 (cyan) (scale bar = 100 μm). Arrows point to central lacteal (GPR182+, mCherry, red) and submucosal lymphatics (left and right). (C) Immunofluorescence image of a section from small intestine from an ACKR4GFP/GFP mouse, showing ACKR4 expressing cells (GFP, green). Smooth muscle cells are positive for α-SMA (red), and cell nuclei in DAPI (blue), scale bar: 20 μm. (D) Immunofluorescence image of a section from small intestine from an ACKR4GFP/GFP mouse, showing ACKR4 expressing cells (GFP, green) in close proximity to c-kit–positive Cajal cells (red); cell nuclei DAPI (blue), scale bar: 20 μm. (E) Confocal images of colon from an ACKR3GFP/+ − GPR182mCherry/+ mouse, GPR182 (mCherry, red), ACKR3 (GFP, green), CD31 (pink), and Lyve-1 (cyan) (scale bar = 100 μm). (F) Confocal images of colon from an ACKR4GFP/+ − GPR182mCherry/+ mouse, GPR182 (mCherry, red), ACKR4 (GFP, green), CD31 (pink), and Lyve-1 (cyan); arrows indicate the lymphatic vessels (scale bar = 100 μm). (G) Immunofluorescence image of a section of colon of an ACKR4GFP/GFP mouse, showing cells expressing ACKR4 (GFP, green) and α-SMA (red); cell nuclei DAPI (blue), scale bar: 20 μm. (H) Immunofluorescence image of a colon section from an ACKR4GFP/GFP mouse, showing ACKR4 expressing cells (GFP, green) in close proximity to c-kit–positive Cajal cells (red); cell nuclei DAPI (blue), scale bar: 10 μm. GFP, green fluorescent protein. https://doi.org/10.1371/journal.pbio.3002111.g006

Use of ACKR-semi-specific chimeras to reveal scavenging activity To study the surface expression of the 3 receptors and their internalization capacity, we generated chimeric chemokines for each receptor; as due to the promiscuity of the chemokine system, specific ligands were missing. We had previously generated the chimeras CXCL11_12 and CCL25_19 for ACKR3 and ACKR4, respectively (36;48). The chimeras consist of the N-terminus of CXCL11 or CCL25 and the body of CXCL12 or CCL19. Both CXCL11 and CXCL12 also bind CXCR3 and CXCR4, respectively. CCL25 and CCL19 are specific ligands for CCR9 and CCR7, respectively. We therefore sought to design and produce a GPR182-specific chimeric chemokine. Considering that the spectrum of chemokines bound by GPR182 has been suggested to be generally broader than for other ACKRs, we selected the N-terminus of human CXCL11, composed of 8 amino acids, and the body of murine CCL20. We specifically chose to hybridize a chemokine of the human CXC family to one of the murine CC family to reflect the broad scavenging ability of this novel receptor. The chimeric chemokine was named CXCL11_20 (S6 Fig). Despite the marked differences in their primary amino acid sequences, the 3 chimeras shared biophysical properties, including the disulfide bridges, molecular weight range (9.6 to 13.2 kDa), isoelectric points (pI 9.5 to 10.5), and they eluted similarly between 32% and 40% acetonitrile from C18 reverse phase columns. The specificity of CXCL11_20 for GPR182 was tested by flow cytometry (Fig 7A). We incubated CXCL11_20 labelled with Atto565 with mouse 300.19 pre-B cells transfected with ACKR1, ACKR2, ACKR3, ACKR4, and GPR182 (expressed using a T2A-GFP system) at 4°C to measure binding and at 37°C to measure uptake. At 4°C, plasma membranes are stiff and receptor endocytosis is blocked; therefore, only surface receptors are available for binding. GPR182 efficiently bound and took up the chimeric construct. Virtually, no binding and uptake was observed in cells transfected with ACKRs, except for marginal uptake from ACKR2. PPT PowerPoint slide

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TIFF original image Download: Fig 7. Chimeric chemokines may be used to detect ACKR expression. (A) In vitro binding and uptake assay: 300.19 pre-B cells expressing ACKRs or GPR182 were incubated with CXCL11_20 to test for binding (4°C, blue bars) or uptake (37°C, red bars). MFI was normalized to GFP expression (n = 3, error bars ± SD). (B) Confocal images of uptake of IV-injected CXCL11_20 into endosomes (green) in spleen sinusoids from GPR182mCherry/+ mouse (mCherry, red). Yellow shows colocalization in merged images, CD21/35 (blue) (scale bar = 30 μm). (C) Confocal images of spleen sinusoids following coinjection of CCL25_19-Atto488 (green) and CXCL11_20-AF647 (red) GPR182mCherry/+ heterozygous mice (scale bar = 10 μm). (D) Uptake of the same chemokines by GPR182-KO (GPR182mCherry/mCherry) mice (scale bar = 10 μm). (E) CXCL11_20 in vivo binding and uptake by GPR182-proficient (red histogram) and GPR182-deficient (blue) spleen endothelial cells (gated on CD45− CD31+). (F) Displacement binding of CXCL11_20 (black), CXCL11_12 (green), and CCL25_19 (red) on GPR182-expressing 300.19 pre-B cells (n = 3 ± SD). (G) Confocal image of a liver section from a wild-type mouse following triple coinjection of CXCL11_20 (red), CCL25_19 (blue), and CXCL11_12 (green). Purple shows colocalization between blue and red. Arrows point to central veins taking up CXCL11_12 via ACKR3. Dashed circles highlight areas of ACKR4 and GPR182 expression and coexpression (scale bar = 100 μm). FCS files and gating strategies are available in FlowRepository (Fig 7E). S4_Data (related to Fig 7A): https://doi.org/10.6084/m9.figshare.22362415; S5_Data (related to Fig 7F): https://doi.org/10.6084/m9.figshare.22362403. ACKR, atypical chemokine receptor; GFP, green fluorescent protein; MFI, mean fluorescence intensity. https://doi.org/10.1371/journal.pbio.3002111.g007 The generation of chimeras allowed us to study the activity of each receptor in situ. To validate the new tool, we injected CXCL11_20 into a GPR182mCherry/+ reporter mouse (Fig 7B). We isolated, fixed, and processed spleens for confocal imaging, 30 minutes later. We observed exact colocalization of endosome-like structures containing the chimera in cells expressing GPR182-mCherry. To further test the specificity of CXCL11_20 for GPR182 in vivo, we injected CXCL11_20 labeled with AF647, together with the chimera for ACKR4, CCL25_19-Atto488, as a control, in GPR182mCherry/+ (heterozygous (GPR182+/−); Fig 7C) and GPR182mCherry/mCherry (both alleles replaced by mCherry, therefore KO (GPR182−/−); Fig 7D) mice. Spleens were isolated, fixed, and processed for imaging, 30 minutes postinjection. As expected, both chemokines were taken up in heterozygous animals. However, GPR182-KO animals failed to take up CXCL11_20 (Fig 7C and 7D), and only endosomes containing CCL25_19 were identified, which indicate uptake by ACKR4, which is also expressed in these splenic sinusoids. Alternatively, endothelial cells were isolated from spleens by enzymatic digestion, CD45+ cells depleted, and analyzed by flow cytometry by gating on CD45− CD31+ cells. The CXCL11_20 fluorescence could only be detected with cells derived from GPR182mCherry/+ mice but not in GPR182mCherry/mCherry (Fig 7E). Taken together, these results establish CXCL11_20 as a GPR182-specific chemokine. The recent finding that GPR182 can broadly scavenge chemokines prompted us to test the ability of GPR182 to bind and scavenge CXCL11_12 and CCL25_19, which we had previously produced for ACKR3 and ACKR4, respectively. We performed competition binding experiments by incubating transfected 300.19 pre-B cells with a low concentration (5 nM) of fluorescent chimeric chemokine, which was outcompeted with increasing concentrations of its unlabeled version. GPR182 bound CXCL11_20 and CXCL11_12 with a calculated Kd of around 60 nM and 65 nM, respectively (Fig 7F). However, the affinity of CXCL11_12 for GPR182 was about 60-fold lower than the reported Kd for ACKR3 [48]. In case of CCL25_19, the affinity for GRP182 was even lower with a Kd around 300 nM, which is about 300 times less than the affinity of the chimera for ACKR4 [36].

Chimera coinjection reveals polarized expression of GPR182 and ACKR4 in the liver Imaging organs such as the liver using fluorescent reporter animals can pose a challenge given by the endogenous autofluorescence, especially in the GFP channel. Therefore, our chimeric ACKR ligands became useful to overcome this. Taking advantage of the fact that the affinity of GPR182 for CCL25_19, the chimera designed for ACKR4, and CXCL11_12, the chimera designed for ACKR3, was relatively low, and that CXCL11_20 is specific for GPR182, we intravenously coinjected the 3 chimeras labeled in different colors (Atto488, Atto565, or AF647) into a C57BL/6 mouse. After 30 minutes, livers were perfused and processed. As expected, the chimeras were found in seemingly endosomal structures within sinusoidal endothelial cells (Fig 7G). Interestingly, we noticed an apparently inverse gradient of uptake of either CCL25_19 or CXCL11_20, suggesting an inverse relationship of expression between ACKR4 and GPR182. More specifically, as shown within the white dashed circles, the sinusoids immediately surrounding central veins primarily displayed uptake of CCL25_19 (Fig 7G, in blue). In more distant sinusoids, we could find primarily “magenta” endosomes, which suggest concomitant uptake of CCL25_19 and CXCL11_20 (Fig 7G, in red). Further away, we observed mainly red endosomes, indicative of CXCL11_20 uptake. In other words, this suggests that ACKR4 is expressed in the sinusoids closest to central veins; ACKR4 and GPR182 are coexpressed (and are similarly active) in sinusoids that are found slightly further away; and GPR182 alone decorates the outermost sinusoids. The similar biophysical properties of the chimeras suggest a similar diffusion, further supporting different expression of the receptors in blood vessels. Inspecting ACKR4-eGFP expression in liver revealed weak signal and considerable autofluorescence background (S7A Fig); therefore, the signal was amplified using an anti-eGFP antibody (S7B Fig). The pattern of ACKR4 liver expression recapitulated the observations of CCL25_19 binding, with higher expression near central veins (S7B Fig) and diminishing towards the edges of lobules (S7B Fig). CXCL11_12 (Fig 7G, green) was more evenly dispersed but was found in endosomes in central veins (white arrows). Taken together, these data highlight that chimeric chemokines can be a valuable tool to study levels of active ACKR expression and their function in health and disease.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002111

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