Stimulation of Proliferation and Migration of Mouse Macrophages by Type B CpG-ODNs Is F-Spondin and IL-1Ra Dependent

Abstract

Macrophage proliferation and migration are important for many facets of immune response. Here we showed that stimulation of macrophages with type B CpG oligodeoxynucleotides (CpG-B ODNs) such as CpG-ODN 1668 increased the production of anti-inflammatory cytokine interleukin 1 receptor antagonist (IL-1Ra) in a TLR9- and MyD88-dependent manner. The CpG-B ODNs-induced IL-1Ra increased macrophage migration and promoted macrophage proliferation by down-regulating the expression of a cell cycle negative regulator, p27 to increase cell population in the S phase. The induction of IL-1Ra by CpG-B ODNs was F-spondin dependent. Knockdown of F-spondin and IL-1Ra decreased CpG-B ODNs-induced macrophage migration whereas overexpression of IL-1Ra increased migration of those cells. These findings demonstrated novel roles for F-spondin and IL-1Ra in CpG-B ODNs-mediated cell proliferation and migration of macrophages.

Fig 1. Type B CpG ODNs increase IL-1Ra expression in a TLR9- and MyD88-dependent manner.

(A) RAW264.7 cells were treated with or without 1 μM of various ODNs and their controls for 6 h and the mRNA expression of IL-1Ra was analyzed by RT-PCR. The gel shown is the representative of three independent experiments (means ± SEM). (B) IL-1Ra production of cells treated with/without 6 μM ODN1668 for 24 h was measured by ELISA; ***, P < 0.001. (C) Cells were treated with or without 20 μM chloroquine in complete medium for 1 h prior to ODN treatment (1 μM). Six hours later, cells were harvested for RT-PCR analysis. (D) THP-1 cells were treated with 2 μM ODN2006 for the times indicated and assayed for IL-1Ra mRNA expression. (E-F) THP-1 and MyD88 deficient THP-1 (defMyD) stable cell lines were treated with or without 2 μM ODN2006 for 24 h. The expression of IL-1Ra was analyzed by both RT-PCR (E) and western blotting (F).

Fig 2. ODN1668 increases IL-1Ra expression via F-spondin.

(A) RAW264.7 cells were stimulated with recombinant F-spondin proteins (rF-spondin) for 24 h and IL-1Ra production was measured by ELISA; **, P < 0.01. (B) RAW264.7 cells were transfected with either Myc-tagged F-spondin plasmid (pF-spondin) or a control vector (VC), and incubated for 24h. The success of transfection was verified by western blotting analysis of F-spondin and Myc expression. The IL-1Ra production was measured by ELISA 48h after transfection; **, P < 0.01. (C) Effect of F-spondin knockdown on ODN1668-induced IL-1Ra expression. Knockdown of F-spondin was verified by RT-PCR analysis. Wild-type RAW264.7 cells (RAW^WT) and RAW264.7 cells stably transfected with F-spondin shRNA (RAW^{spondin-shRNA}) or scrambled shRNA (RAW^Mock-shRNA) were incubated with or without 1 μM ODN1668 for the indicated times. RT-PCR was then performed to analyze IL-1Ra expression. β-actin was used as a loading control. (D) Bone marrow derived macrophages were transfected with scrambled siRNA or siRNA for F-spondin gene. After cells were attached to the plate, media were removed and fresh media containing 6 μM ODN1668 were supplied. The expressions of F-spondin and IL-1Ra were examined after 1d treatment. Similar results were obtained in two independent experiments. (E) The IL-1Ra production of cells in (D) was measured by ELISA; **, P < 0.01. Data shown are average of three independent experiments.

Fig 3. Overexpression of IL-1Ra increases RAW 264.7 cell proliferation through increasing S phase cell population.

(A) RAW264.7 cells treated with or without 5 μg/ml recombinant F-spondin protein (rF-spondin) were cultured in complete media for 48 h and assayed for viability; ***, P < 0.001. (B) RAW264.7 cells treated with or without 1 μM ODN1668 and IL-1Ra overexpressing stable cell line (RAW^IL-1Ra) were cultured in complete media for 48 h and assayed for viability; ***, P < 0.001. Overexpression of IL-1Ra in RAW^IL-1Ra was confirmed by RT-PCR. (C) Wild-type RAW264.7 cells (WT), RAW264.7 cells stably transfected with empty vector (VC), and RAW^IL-1Ra cells (RA) were plated at the same density and cultured in complete media for 48 h. Cell cycle analysis was performed by flow cytometry and the data were analyzed using ModFit software. Results shown are representative of three independent experiments; **, P < 0.01. (D) The expression of cell cycle-related protein in RAW^VC and RAW^IL-1Ra cells were examined by western blotting. (E) Bone marrow derived macrophages (BMMs) were transiently transfected with a control vector (VC) or IL-1Ra plasmid (RA). The expressions of IL-1Ra and p27 were examined 24h after transfection. (F) Western blotting analysis of IL-1Ra and p27 expressions in BMMs transfected with scrambled siRNA or siRNA for IL-1Ra gene. Cells were incubated with 6 μM ODN1668 for 24h before harvest.

Fig 4. ODN1668 promotes macrophage migration via the F-spondin/IL-1Ra signaling pathway.

Trajectories of cells in response to different treatments were measured by time-lapse microscopy and displayed in diagrams drawn with the initial point of each trajectory placed at the origin of the plot. Serial phase-contrast images were obtained every 15 min for 22 h by time-lapse microscopy. Trajectories (upper panel) and migration velocity (lower panel) of cells were displayed after 22 h of time-lapse. Results of migration velocities represent data of 20 cells (means ± SEM). (A) RAW264.7 and J774A.1 cells were treated with or without 6 μM ODN1668 or 6 μM GpC ODN1668 (ODN1668GC). (B) RAW264.7 and J774A.1 cells were treated with or without 2 μg/ml recombinant F-spondin protein (rF-spondin). (C) RAW^Mock-shRNA and RAW^{Spondin-shRNA} stable cell lines were treated with or without 6 μM ODN1668. (D) The trajectory and migration velocity of untreated RAW^VC and RAW^IL-1Ra stable cell lines were compared. (E) RAW264.7 cells were transfected with scrambled siRNA or siRNA for IL-1Ra gene. After cells were attached to the plate, media were removed and fresh media containing 6 μM ODN1668 were supplied. The transfection efficiency was verified by western blotting. *, P < 0.05, ****, P < 0.0001, N.S., not significant.

Fig 5. Knockdown of F-spondin and IL-1Ra diminishes the effect of ODN1668 on migration of bone marrow derived macrophages (BMMs).

BMMs were transfected with scrambled siRNA or siRNA for IL-1Ra and/or F-spondin gene. After cells were attached to the plate, media were removed and fresh media containing 6 μM ODN1668 were supplied. Trajectories (upper panel) and migration velocity (lower panel) of cells were displayed after 22 h of time-lapse as described in the Materials & Methods section. **, P < 0.01, ****, P < 0.0001.