Macrophages from Ltb4r2−/− mice exhibit reduced chemotaxis. Bone-marrow-derived macrophages (BMDMs) were isolated from WT and Ltb4r2−/− mice and unpolarized (M0) or polarized to M2-like and M1-like states. A, schematic representation of experimental design; (B) iNOS+ cells as a percentage of F4/80+ CD11b+ cells as determined by flow cytometry; (C) CD206+ cells as a percentage of F4/80+ CD11b+ cells as determined by flow cytometry; (D) principal component analysis plot of RNA sequencing data; (E) Volcano plot showing differentially expressed genes by p value in M0 macrophages (each dot represents a distinct gene); (F) Volcano plot showing differentially expressed genes by p value in M2-polarized macrophages; (G) Volcano plot of differentially expressed genes by p value in M1-polarized macrophages; (H) Enrichr Gene Ontology pathway analysis of differentially expressed genes in M1-polarized macrophages, ordered by p-value; (I) relative expression of Ccl5 and Lgals3 in M1-like macrophages from WT and Ltb4r2−/− mice; (J) representative images of macrophage migration in transwell plates (left panels) and respective quantification (right panel). Arrows indicate macrophages (blue) in left panel images. Scale bar = 500 μm; (K) quantification of migrated macrophages after treatment with vehicle (V) or 6 μg LY255283 (LY). All data are presented as mean ± SEM and each data point represents an independent biologic replicate from different animals. For statistics, unpaired two-tailed t tests were used for comparison of two values, and 1-way ANOVA with Tukey post-test for more than two comparisons.

Morpholino oligonucleotide against ltb4r2a in zebrafish impairs macrophage migration.A, qRT-PCR for ltb4r2a and ltb4r2b during different ages of zebrafish. N = 3 biological replicates per time point. B, schematic representation of tail cut injury in Tg(mpeg:eGFP)gl22 zebrafish larvae in which macrophages are labeled green. C, representative images of macrophages (green) and DNA (magenta) (left panels) following injection of morpholino oligonucleotides (MOs) against ltb4r2a and ltb4r2b, and corresponding quantification of macrophage number at the site of injury (right panels). Scale bar = 100 μm; D, representative images of macrophages (green) in Tg(mpeg:eGFP)gl22 zebrafish larvae yolk sac (left panels) and quantification of macrophage numbers (right panel). Scale bar 100 μm. E, representative images of macrophages (green) and DNA (magenta) in zebrafish larvae injected with indicated MOs (left panels) and quantification of macrophage number at the site of injury (right panel). Scale bar = 100 μm. F, representative images of 1 dpf zebrafish embryos following injection with GFP mRNA with or without the ltb4r2a MO. Scale bar = 500 μm. G, representative images of macrophages (green) and DNA (magenta) in zebrafish larvae injected with indicated MOs and mRNAs (left panels) and quantification of macrophage number at the site of injury (right panel). Scale bar = 100 μm. H, representative images of macrophages (green) and DNA (magenta) in zebrafish larvae treated with vehicle or indicated concentrations of LY255283 (left panels) and quantification of macrophage number at the site of injury (right panel). Scale bar = 100 μm. I, representative images of macrophages (green, indicated by black arrowheads) in Tg(mpeg:eGFP)gl22 zebrafish larvae yolk sac following treatment with with vehicle or indicated concentrations of LY255283 (left panels) and quantification of macrophage numbers (right panel). Scale bar = 100 μm. Data are presented as mean ± SEM; Each data point represents an independent biological replicate from different animals. For statistics, unpaired two-tailed t-tests were used for comparison of two values, and 1-way ANOVA with Tukey post-test for more than two comparisons.

Morpholino oligonucleotide against ltb4r2a in zebrafish impairs macrophage migration during β-cell and liver inflammation.A, schematic representation of the β-cell injury model using Tg(ins:NTR)^S950;Tg(mpeg:eGFP)^gl22 zebrafish expressing nitroreductase (NTR) in β cells and green fluorescent protein in macrophages. Double-transgenic larvae were treated with metronidazole (MTZ) for 6 h, followed by imaging of islets. B, representative images of macrophages (green) and insulin (red) following injection of morpholino oligonucleotide (MO) and treatment with MTZ, as indicated (left panels), and quantitation of macrophage number within islets (right panel). Scale bar = 10 μm. C, schematic representation of the liver injury model using Tg(mpeg:eGFP)^gl22 zebrafish expressing green fluorescent protein in macrophages. Transgenic larvae were injected with or without MO at 0 to 1 h post fertilization, then incubated in 5% egg solution in egg water as indicated. D, representative images of macrophages (green) and DNA (magenta) and quantification of macrophage number in the liver. The dotted line indicates the outline of the liver parenchyma and arrowheads indicate macrophages (green) within the liver parenchyma. Scale bar = 100 μm. Data are presented as mean ± SEM; and each data point represents an independent biological replicate from different animals. For statistics, unpaired two-tailed t-tests were used for comparison of two values, and 1-way ANOVA with Tukey post-test for more than two comparisons.

Ltb4r2−/− mice exhibit impaired macrophage migration following LPS-induced inflammation.A, schematic representation of LPS injury model in mice. WT and Ltb4r2−/− mice were injected with lipopolysaccharide (LPS), followed by quantitation of peritoneal macrophage number (by flow cytometry) and number of macrophages infiltrating the liver (by immunohistochemistry). B, representative flow cytometric dot plots of peritoneal F4/80+ cells (macrophages) (left panels) under the indicated conditions and corresponding quantitation (right panel). C, quantitation (from flow cytometry analysis) of F4/80+ and Cd11c+ cells from spleen in WT and Ltb4r2−/− mice following LPS injection. D, representative liver section images of Galectin 3+ macrophages (blue, indicated by arrows) under the indicated conditions (left panels) and quantification of macrophage number in liver tissue (right panel). Scale bar = 100 μm. Data are presented as mean ± SEM; ∗p < 0.05, and each data point represents an independent biological replicate from different animals. For statistics, 1-way ANOVA with Tukey post-test was used for comparisons.