GPR182 is a broadly scavenging atypical chemokine receptor influencing T-independent immunity

Abstract

Immune responses highly depend on the effective trafficking of immune cells into and within secondary lymphoid organs (SLOs). Atypical chemokine receptors (ACKRs) scavenge chemokines to eliminate them from the extracellular space, thereby generating gradients that guide leukocytes. In contrast to canonical chemokine receptors, ACKRs do not induce classical intracellular signaling that results in cell migration. Recently, the closest relative of ACKR3, GPR182, has been partially deorphanized as a potential novel ACKR. We confirm and extend previous studies by identifying further ligands that classify GPR182 as a broadly scavenging chemokine receptor. We validate the "atypical" nature of the receptor, wherein canonical G-protein-dependent intracellular signaling is not activated following ligand stimulation. However, β-arrestins are required for ligand-independent internalization and chemokine scavenging whereas the C-terminus is in part dispensable. In the absence of GPR182 in vivo, we observed elevated chemokine levels in the serum but also in SLO interstitium. We also reveal that CXCL13 and CCL28, which do not bind any other ACKR, are bound and efficiently scavenged by GPR182. Moreover, we found a cooperative relationship between GPR182 and ACKR3 in regulating serum CXCL12 levels, and between GPR182 and ACKR4 in controlling CCL20 levels. Furthermore, we unveil a new phenotype in GPR182-KO mice, in which we observed a reduced marginal zone (MZ), both in size and in cellularity, and thus in the T-independent antibody response. Taken together, we and others have unveiled a novel, broadly scavenging chemokine receptor, which we propose should be named ACKR5.

Keywords: GPR182; atypical chemokine receptor; chemokine; marginal zone; signal transduction.

Figure 1

GPR182 binds and internalizes a broad variety of chemokines with low affinity. (A) 300.19 pre-B cells expressing mGPR182 T2A GFP (left panels) or hGPR182 T2A GFP (right panels) were incubated (together with parental GFP negative 300.19 cells) with 200 nM fluorescently labelled human or mouse chemokines (as indicated in each panel) at 37°C for 45 min and uptake was measured by FACS. Panels are grouped for shared binding and uptake by other ACKRs as indicated. Shown is one representative experiment out of 5 independent determinations performed. (B, D) Competition binding at 4°C or (C, E) scavenging at 37°C was measured by incubating 300.19 cells expressing mGPR182 (B, C) or hGPR182 (D, E) with a fixed concentration of fluorescent chemokine (3 nM) and increasing concentrations of its unlabeled counterpart. (N = at least 3 independent experiments, performed in duplicates. Error bars ± SD).

Figure 2

GPR182 is a chemokine scavenger. (A) Surface rendering of internalized 200 nM CXCL12-AT700 or (A) 200 nM CCL28-AF647 (B) (red) in HEK293 cells stably transfected with mGPR182 T2A GFP (left panels, green) or hGPR182 T2A GFP (right panels, green). Scale bars = 5 to 7 μm. (C) Remaining hCXCL13 or (D) remaining mCXCL13 levels in the cell culture medium following overnight incubation with either HEK293 WT or HEK293 expressing mGPR182 or hGPR182. ELISA measurements were normalized to CXCL13 levels in supernatants of HEK293 WT cells. (N = at least 2 independent experiments, performed in duplicates. Error bars ± SD. Unpaired two-tailed t-test *p<0.05 **p<0.01 ***p<0.001). (E, F) Degradation of hCXCL13 (E) by HEK293 cells expressing mGPR182 or hGPR182. Degradation of mCXCL13 (F) by HEK293 cells expressing mGPR182 or primary mouse LECs, in absence or presence of 50 nM Bafilomycin A1 (Baf A1). Shown are western blots of corresponding cell lysates. GAPDH was used as loading control. (G, H) Confocal images illustrating co-localization of CXCL11_20 (red) and Rab5 (top panels) or Lamp1 (bottom panels) in HEK293 cells expressing mGPR182 (G) or hGPR182 (H). Panels on the right are magnifications of each image. White arrows point to co-localization of GPR182 and the markers. Scale bars = 3μm [(G) top left panel, (H) left panels], 2 [(G) top left panel, (H) left panels], 2μm [(G) bottom left panel], 1μm (right panels).

Figure 3

GPR182-chemokine interactions do not induce canonical signaling. (A) Stimulation of intracellular free calcium raise by 50 nM CXCL12 (left) and CCL19 (right) in 300.19 pre-B cells expressing cognate canonical chemokine receptors (CXCR4 (left) and CCR7 (right), black lines) and mGPR182 (red lines) or hGPR182 (green lines). CaCl₂ + Ionomycin were added for comparison. (N = average of 15 cells, performed in duplicate at least twice). (B) ERK phosphorylation was measured by western blot (upper panel) upon stimulation with 50 nM CXCL11 (X11) or CXCL12 (X12) using 300.19 pre B cells expressing CXCR3, CXCR4, mGPR182 or hGPR182 (from left to right). Bar charts show quantification of normalized (loading control) pERK signal intensity from western blots (relative to unstimulated controls). N = 2 independent experiments performed in duplicates. Error bars ± SD. Dashed line represents threshold. Unpaired two-tailed t-test *p<0.05 **p<0.01. (C–F) Analyses of chemokine-induced responses in HeLa cells expressing mGPR182 (C, E) and hGPR182 (D, F). Recruitment of LgBIT-miniG_αi to mGPR182-nLuc11 (C) or hGPR182-nLuc11 (D) and G protein activation measured as dissociation of Gα-nLuc from Gβγ-cpVenus in cells expressing mGPR182 (E) or hGPR182 (F) stimulated with 300 nM chemokine. HeLa cells expressing hCCR6-nLuc11 (black dots) served as positive controls upon stimulation with hCCL20. (N = 3, mean ± SEM).

Figure 4

GPR182 requires GRK and β-arrestins for constitutive internalization and scavenging, but not its C-terminus. (A) Confocal images illustrating expression of tagged mGPR182-venus (green) in HEK293 WT cells (upper panels) or in HEK293 cells lacking β-arrestin expression (β-arrestin 1,2 KO) (lower panels), with enlargements (right) showing surface rendering of endosomal-like structures in WT cells and of receptor surface expression in KO cells (nuclei in blue, DAPI). Scale bars = 5 to 10 μm. (B) Quantification of integrated fluorescence intensities measured by confocal microscopy (measured with ImageJ) of internalized CXCL11_20-ATTO565 (200 nM) in HEK293 cells expressing mGPR182-venus. From left: cells deficient for β-arrestins (KO black column), cells expressing β-arrestin 1 (squares, blue column), β-arrestin 2 (upward triangle, red column), combined β-arrestin 1 and 2 (downward triangles, purple column), and HEK293^WT (black circles, green column). Data from one representative experiment, 3 fields of view were analyzed with at least 5 cells per field of view measured. Error bars ± SD. Unpaired two-tailed t-test, **p<0.01 ***p<0.001 ****p<0.0001. (C–F) Confocal images illustrating expression of mGPR182-venus (green), CXCL11_20 uptake (red), plasma membrane staining (cyan) and DAPI nuclear staining (blue) in HEK293 WT (C), GRK 2/3 KO (D), GRK 5/6 KO (E), and GRK 2/3/5/6 KO cells (F). Middle panels show DAPI with mGPR182-venus (blue/green), or DAPI with CXCL11_20 (blue/red). Bottom panels depict 3D rendering showing chemokine internalization in endosomal like structures. Scale bars = 5μm (C, D, F) and 4μm (E). (G) Confocal images showing expression of mGPR182-venus (green, left), or mGPR182ΔC-venus (green, right), CXCL11_20 uptake (red), plasma membrane staining (cyan) and DAPI nuclear staining (blue) in HEK293 cells. Middle panels show DAPI with mGPR182-venus (blue/green), or DAPI with CXCL11_20 (blue/red). Bottom panels depict 3D rendering showing chemokine internalization in endosomal like structures. Scale bars = 5μm (top and middle panels), 2μm (bottom, left) 3μm (bottom, right). (H) MFI of CXCL11_20 uptake relative to the fluorescence of Venus measured by FACS in HEK293^WT cells expressing GPR182-Venus (white bar) or mGPR182ΔC-venus (green bar), β-arrestin 1,2^KO cells expressing mGPR182ΔC-venus (β-Arr KO mGPR182ΔC, hatched green bar). MFI of surface bound CXCL11_20 was measured by incubating cells with chemokine at 17°C and was subtracted from MFI of uptake at 37°C. N = 3. Error bars ± SD. Unpaired two-tailed t-test, ***p<0.001 ****p<0.0001.

Figure 5

CXCL13 and CCL28 are GPR182-specific ligands. MFI of binding (4°C, blue columns) and uptake (37°C, red columns) of 100 nM hCXCL13 (A) and mCCL28 (B) by 300.19 pre-B cells expressing either ACKR1, ACKR2, ACKR3, ACKR4, mGPR182 or hGPR182 (T2A GFP). The background MFI of chemokines incubated with untransfected 300.19 pre-B cells was subtracted. Pooled data from at least 2 independent experiments, error bars ± SD. Unpaired two-tailed t-test, **p < 0.01, ****p < 0.0001. (C, D) Confocal images showing uptake in spleen sinusoids of mice co-injected i.v. (2.5 μM each) with fluorescently labelled mCXCL13-AF647 (green) (C) and CXCL11_12-AF488 (red) (scale bar = 20μm) or mCCL28-AF647 (green) (D) and CXCL11_12-AF488 (red) (scale bar = 5μm) into GPR182^+/- and GPR182^-/- mice (mCherry GPR182 reporter expression in grey). White arrows indicate areas of colocalization between red and green (yellow). Representative data from at least two independent experiments.

Figure 6

GPR182 regulates serum chemokine levels. (A) Serum levels of CXCL13 in C57B6 (open circles), GPR182^+/- (black) and GPR182^-/- (red) mice. (B) Serum level of CCL28 in C57B6 (open circles), GPR182^+/- (black) and GPR182^-/- (red) animals. (C) Serum levels of CXCL12 in C57B6 (open circles), GPR182^+/- (black circles), GPR182^-/- (red circles), GPR182^+/- treated with CCX771 (black triangles), and GPR182^-/- treated with CCX771 (red triangles) animals. Serum level of CCL19 (D), CCL20 (E), CCL21 (F) in C57B6 (open circles), GPR182^+/- (black circles), GPR182^-/- (red circles), ACKR4^-/- (green diamonds), GPR182^-/–ACKR4^-/- (open diamonds) animals. (G–J) Interstitial chemokine levels: CXCL12 (G) in spleen and (H) in LN, CXCL13 (I) in spleen and (J) in LN. Chemokine levels were normalized to total protein content. (N=3-16 per group. Error bars means ± SD (means thick horizontal line). Unpaired two-tailed t-test *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns not significant).

Figure 7

GPR182^-/- mice have a reduced marginal zone. (A) Representative FACS plots indicating the percentage of MZBs (CD21^Hi CD23^- cells gated on CD19⁺ B220⁺) in GPR182^+/- (left) and GPR182^-/- (right) mice. (B) Quantification of multiple measurements, means ± SD (N = at least 7/group). (C) Representative confocal images of CD21/35 staining (green) to show MZ, found at the border with the marginal sinus, MAdCAM-1 (red) (left, scale bar = 200μm). Right panels: enlargements with measurement bar used for quantification (right, scale bar = 50μm). (D) Quantification of MZ thickness (N=5 spleens/group, with at least 5 follicles/spleen analyzed). Mean fluorescence intensity (MFI) of receptor expression on MZBs CXCR4 (E) and CXCR5 (F); and on FoBs CXCR4 (G) and CXCR5 (H), CD29 expression (integrin β1 chain) on MZB (I) (N= at least 7 mice/group). (J) Representative confocal images of deposition of TNP-Ficoll-fluorescein (green) on spleen FDCs (CD21, red) within the follicle (lined by MAdCAM-1⁺ marginal sinus, cyan), in GPR182^+/- (left) and GPR182^-/- (right) mice, 3h post-injection. (K) Quantification of TNP-Ficoll-fluorescein deposition on CD21⁺ FDCs of GPR182^+/- (black circles) and GPR182^-/- (red circles) mice (N=5 mice/group). (L) Levels of TNP-specific IgM at day 4 post immunization in serum of GPR182^+/- (black circles) and GPR182^-/- (red circles) mice measured by ELISA (N=7 mice/group). Error bars means ± SD, mean thick horizontal line. Unpaired two-tailed t-test: *p<0.05, ***p<0.001, ****p<0.0001, ns not significant).