GPR35 and mediators from platelets and mast cells in neutrophil migration and inflammation

Abstract

Neutrophil recruitment from circulation to sites of inflammation is guided by multiple chemoattractant cues emanating from tissue cells, immune cells, and platelets. Here, we focus on the function of one G-protein coupled receptor, GPR35, in neutrophil recruitment. GPR35 has been challenging to study due the description of multiple ligands and G-protein couplings. Recently, we found that GPR35-expressing hematopoietic cells respond to the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA). We discuss distinct response profiles of GPR35 to 5-HIAA compared to other ligands. To place the functions of 5-HIAA in context, we summarize the actions of serotonin in vascular biology and leukocyte recruitment. Important sources of serotonin and 5-HIAA are platelets and mast cells. We discuss the dynamics of cell migration into inflamed tissues and how multiple platelet and mast cell-derived mediators, including 5-HIAA, cooperate to promote neutrophil recruitment. Additional actions of GPR35 in tissue physiology are reviewed. Finally, we discuss how clinically approved drugs that modulate serotonin uptake and metabolism may influence 5-HIAA-GPR35 function, and we speculate about broader influences of the GPR35 ligand-receptor system in immunity and disease.

Keywords: GPCRs; chemotaxis; lipid mediators; mast cells; neutrophils; platelets.

Figure 1.. Mouse Gpr35 transcript abundance in neutrophils and other immune cells and of serotonin receptors in neutrophils.

Data are from Immgen.org. The neutrophil (GN) data are for CD11b⁺Ly6G⁺ immature cells in the BM, unstimulated cells from spleen, and cells recruited to the peritoneal cavity 18 hours after thioglycolate (1ml, 3%) injection. See Immgen.org for detailed descriptions of the other cell types.

Figure 2.. Summary diagram of GPR35 ligands and functions.

The overall structure of human GPR35 with the agonist Lodoxamide is shown (PDB:8H8J). ‘Endogenous ligands’ indicates molecules that can be generated by physiological pathways; evidence for reduced in vivo GPR35 function under conditions of reduced metabolite production has so far only been reported for 5-HIAA. In addition to DHICA, several other tyrosine metabolites have activity in vitro on GPR35 in the μM to mM range. ‘Environmental/Diet’ refers to molecules produced by plants or microbes. While we have tried to be comprehensive, the list of molecules with suggested agonist or antagonistic activity on GPR35 may not be complete. See text for details and references.

Figure 3.. Selective GPR35 agonism by 5-HIAA.

(A, B) TGFα shedding assay with mGPR35 and chimeric Gαq proteins in HEK293 ΔGαq/11/12/13 cells that express the alkaline phosphatase (AP)-TGFα membrane construct. Gq activation leads to AP-TGFα shedding and allows GPCR activation to be measured based on AP activity in the culture supernatant. Gαq/i1 reports Gαi coupling, Gαq/13 Gα13 coupling, and Gαq/o Gαo coupling. 8Gα refers to cells receiving a mixture of Gαq/s, Gαq/i1, Gαq/i3, Gαq/o, Gαq/z, Gαq/12, Gαq/13, Gαq/16. (A) Response to Lodoxamide, a synthetic GPR35 agonist. (B) Response to 5-HIAA compared to Lodoxamide. Data are mean ± s.d. (n=3). Experiments were repeated at least twice with similar results. (C-E) Internalization of N-terminally OX56 epitope-tagged WT (C, D) or C-terminally truncated (delta293, E) mGPR35 in M12 cells (C) or WEHI-231 cells (D, E) in response to the indicated stimuli for 30 min at 37°C. M12 and WEHI-231 cell transductions and internalization assays were performed as previously described. Both are mouse B lymphoma cell lines. The del293 mutant lacks the last 13 amino acids of the receptor, including Ser and Thr residues required for β-arrestin recruitment. (F-H) Intracellular FACS for pERK in WEHI-231 B lymphoma cells transduced with mGPR35. Staining of permeabilized cells was with Cell Signaling antibody 197G2 against phospho-ERK1/2 (Thr202/204). Example FACS plots for GPR35-IRES-GFP transduced cells (F) and summary data for untransduced (G) and GPR35-IRES-GFP transduced cells (H) stimulated with diluted carrier (DMSO), 5-HIAA (10nM) or Lodoxamide (20μM) for 10 min.

Figure 4.. Biosynthetic pathway of serotonin and 5-HIAA.

Chemical structure diagrams of L-tryptophan, serotonin and 5-HIAA are shown. Enzyme names are in italics.

Figure 5.. Neutrophil association with platelet coated endothelium in the inflamed mouse omentum.

Neutrophils (Cell trace violet labeled, blue) in the inflamed omentum of a mouse that has all its platelets labeled green (GFP⁺). Image was acquired by intravital 2-photon microscopy two hours after intraperitoneal treatment with thioglycolate. The platelets outline the inflamed endothelium, and many intravascular neutrophils are observed in contact with platelet clusters. Neutrophil and platelet labeling and imaging was performed as in.

Figure 6.. Model of early events in neutrophil recruitment to a site of inflammation where mast cell and platelet activation is occurring.

Panels highlight some of the key molecular mediators acting in the first minutes to hours of recruitment. (A) Mast cells are highlighted as an early source of mediators acting on the endothelium. The extent of their involvement will depend on the tissue and type of stimulus. Other cell types that are not shown such as macrophages will also usually be involved. Serotonin and histamine from mast cells cooperate to promote vascular permeability and Weibel-Palade body mobilization and thus P-selectin (P-sel) display. Other preformed inflammatory mediators such as TNF are also rapidly released. (B) These events are quickly followed by platelet activation, attachment to the endothelium and release of a range of platelet-derived mediators. (C) Neutrophils that first associate with the endothelium and platelets via P-selectin-supported rolling are triggered via chemokines to undergo integrin (αMβ2 and αLβ2) mediated adhesion to the endothelium. (D) Association of adherent, migrating neutrophils with activated platelet clusters is promoted by further Gαi-coupled receptors including GPR35 responding to platelet-derived 5-HIAA. Subsequent extravasation is promoted by the concerted action of multiple chemoattractants including chemokines, PAF and 5-HIAA as well as other mediators and adhesion molecules that are not shown. For simplicity the vascular wall is represented by the endothelium; the associated pericytes, smooth muscle cells and extracellular matrix (ECM) are not shown and nor are additional mediators such as IL-17A that act on these cells and proteases that act on the ECM. Image generated using BioRender.

Figure 7.. Diagram highlighting sites of action of serotonin and 5-HIAA modulating drugs.

Tph1-expressing enterochromaffin cells in the intestine (shaded blue) are a major source of serotonin (5HT). Gut-derived serotonin that reaches circulation is taken up by platelets via the serotonin-selective reuptake transporter (SERT). Serotonin is stored in platelet dense granules but also metabolized via MAO to 5-HIAA. Mast cells express Tph1 and produce serotonin and they also metabolize it to 5-HIAA. Activated platelets and mast cells release both serotonin and 5-HIAA (along with many other metabolites, proteins, and lipids that are not shown). Fluoxetine (Prozac^™) inhibits SERT, and chronic treatment depletes platelets of serotonin and 5-HIAA. Phenelzine inhibits MAO and thus prevents conversion of serotonin into 5-HIAA in platelets and mast cells. Image generated using BioRender.