Wnt signaling modulates macrophage polarization and is regulated by biomaterial surface properties

Abstract

Macrophages are among the first cells to interact with biomaterials and ultimately determine their integrative fate. Biomaterial surface characteristics like roughness and hydrophilicity can activate macrophages to an anti-inflammatory phenotype. Wnt signaling, a key cell proliferation and differentiation pathway, has been associated with dysregulated macrophage activity in disease. However, the role Wnt signaling plays in macrophage activation and response to biomaterials is unknown. The aim of this study was to characterize the regulation of Wnt signaling in macrophages during classical pro- and anti-inflammatory polarization and in their response to smooth, rough, and rough-hydrophilic titanium (Ti) surfaces. Peri-implant Wnt signaling in macrophage-ablated (MaFIA) mice instrumented with intramedullary Ti rods was significantly attenuated compared to untreated controls. Wnt ligand mRNA were upregulated in a surface modification-dependent manner in macrophages isolated from the surface of Ti implanted in C57Bl/6 mice. In vitro, Wnt mRNAs were regulated in primary murine bone-marrow-derived macrophages cultured on Ti in a surface modification-dependent manner. When macrophageal Wnt secretion was inhibited, macrophage sensitivity to both physical and biological stimuli was abrogated. Loss of macrophage-derived Wnts also impaired recruitment of mesenchymal stem cells and T-cells to Ti implants in vivo. Finally, inhibition of integrin signaling decreased surface-dependent upregulation of Wnt genes. These results suggest that Wnt signaling regulates macrophage response to biomaterials and that macrophages are an important source of Wnt ligands during inflammation and healing.

Keywords: Macrophage; Microstructured titanium; Wnt signaling.

Figure 1.

Macrophage ablation reduces peri-implant Wnt signaling in bone. Macrophages were ablated in MaFIA mice by treatment with AP20187 daily for 3d prior to surgery and every other day post-operatively. Control groups only underwent the 3d ablation process, while sham, rough, and rough-hydro groups underwent treatment until sacrifice at 1, 3 or 7d post-surgery (n=6 per group). Expression of Wnt ligand genes in peri-implant tissue was measured using qPCR. Results are normalized to expression of the housekeeping gene Gapdh and presented as fold-change (2^-ΔΔCt). A) Heat map demonstrating the change in Wnt gene expression for with macrophage ablation in Sham, Rough, and Rough-Hydro peri-implant tissue. B) Comparison of Wnt gene expression on Rough-Hydro surfaces in Control (green) and Ablated (orange) animals. p<0.05: A vs. respective Control.

Figure 2.

Macrophages upregulate Wnt ligand expression in response to modified Ti surfaces in vivo. C57Bl/6 mice were instrumented with rough or rough-hydro Ti implants, and peri-implant tissue was collected at 1, 3, or 7d (n=6 per group). Macrophages were isolated by cell sorting using CD11b-APC antibody and subsequent anti-APC magnetic nanobeads. Expression of Wnt ligand genes in peri-implant tissue was measured using qPCR. Results are normalized to expression of the housekeeping gene Gapdh and presented as fold-change (2^-ΔΔCt). p<0.05: # vs. respective Control, $ vs. respective Sham, % vs. respective Rough.

Figure 3.

Macrophages modulate Wnt-related gene expression in response to modified Ti surfaces in vitro. Primary macrophages were grown from whole marrow isolated from C57Bl/6 mice using macrophage colony stimulating factor. Following 7d of culture, macrophages were plated on smooth, rough, or rough-hydro Ti disks, with tissue culture polystyrene (TCPS) serving as control (n=6 per group). Expression of Wnt-related genes was measured using qPCR. Results are normalized to expression of the housekeeping gene Gapdh and presented as fold-change (2^-ΔΔCt). (A) Heat map and volcano plot summarizing changes in macrophage gene expression on smooth, rough, or roughhydro surfaces. (B) Changes in gene expression of Wnt ligands, Wnt receptors, Wnt signaling downstream mediators, and Wnt signaling inhibitors. p<0.05: # vs. respective Control, $ vs. respective Smooth, % vs. respective Rough.

Figure 4.

Treatment with recombinant Wnt ligands alters expression of polarization gene markers. Primary macrophages cultured on TCPS were treated with recombinant Wnt3a, −5a, −5b, −7a, −9b, or −11 for 1d prior to harvest. ELISA was performed to measure pro-(IL-1β, IL-12, TNF-α, CXCL-10) and anti-inflammatory (IL-4, IL-10) protein secretion. Results are normalized to DNA quantity in each sample. p<0.05: # vs. Control, $ vs. Wnt3a, % vs. Wnt5a, & vs. Wnt5b, ^ vs. Wnt7a, and @ vs. Wnt9b.

Figure 5.

Loss of Wnt signaling attenuates inflammatory gene expression in response to classical polarizing stimuli. Macrophageal Wnt secretion was inhibited A) pharmacologically using Wnt-C59 or B) genetically by treating Csf1riCre+; Wls^fl/fl mice with tamoxifen prior to culture of macrophages. Wls expression was used to verify pharmacological inhibition of Wnt secretion, as evidenced by its upregulation upon treatment with Wnt-C59, as well as genetic knockout of Wls, as evidenced by nearly absent expression. Pro-(Il1b, Tnf, Nos2) or anti-inflammatory (Il10, Arg1) genes in response to M1 (lipopolysaccharide) or M2 (Interleukin-4 and −13) stimuli. p<0.05: # vs. M0, $ vs. M1, % vs. M2, A vs. Control of corresponding group.

Figure 6.

Loss of Wnt signaling attenuates inflammatory cytokine production in response to classical polarizing stimuli. Macrophageal Wnt secretion was inhibited A) pharmacologically using Wnt-C59 or B) genetically by treating Csf1r-iCre+; Wls^fl/fl mice with tamoxifen prior to culture of macrophages. Pro-(IL-1β, IL-6, TNF-α) and anti-inflammatory (IL-4, IL-10) cytokines were measured by ELISA and normalized to DNA quantity in each sample (n=6 per group). p<0.05: # vs. M0, $ vs. M1, % vs. M2, A vs. Control of corresponding group.

Figure 7.

Loss of Wnt signaling attenuates inflammatory gene expression in response to Ti surface cues. Macrophageal Wnt secretion was inhibited A) pharmacologically using Wnt-C59 or B) genetically by treating Csf1riCre+; Wls^fl/fl mice with tamoxifen prior to culture of macrophages. Wls expression was used to verify pharmacological inhibition of Wnt secretion, as evidenced by its upregulation upon treatment with Wnt-C59, as well as genetic knockout of Wls, as evidenced by nearly absent expression. Pro-(Il1b, Tnf, Nos2) or anti-inflammatory (Il10, Arg1) genes in response to smooth, rough, or rough-hydro Ti surface modifications. p<0.05: # vs. TCPS, $ vs. Smooth, % vs. Rough, A vs. Control of corresponding group.

Figure 8.

Loss of Wnt signaling attenuates inflammatory cytokine production in response to Ti surface cues. Macrophageal Wnt secretion was inhibited A) pharmacologically using Wnt-C59 or B) genetically by treating Csf1riCre+; Wls^fl/fl mice with tamoxifen prior to culture of macrophages. Pro-(IL-1β, IL-6, TNF-α) and anti-inflammatory (IL4, IL-10) cytokines were measured by ELISA and normalized to DNA quantity in each sample (n=6 per group). p<0.05: # vs. TCPS, $ vs. Smooth, % vs. Rough, A vs. Control of corresponding group.

Figure 9.

Recombinant Wnt ligands rescue impaired Wnt-inhibited macrophage polarization. Macrophages were treated with 500nM Wnt-C59 or equivalent volume of DMSO vehicle and plated on smooth or rough-hydro surfaces. After 2 hours, macrophages were (A) left untreated to observe only surface effects, treated with (B) Wnt3a, −5a, −7a, or −9b on smooth surfaces, or (C) Wnt5b, −7a, −9b, or −11 on rough-hydro surfaces for 24 hours. Pro-(IL-1β, IL-6, IL12(p40), CXCL-10, TNF-α) and anti-inflammatory (IL-4, IL-10) chemokines and cytokines were measured by ELISA and normalized to DNA quantity in each sample (n=6 per group). p<0.05: # vs. TCPS-Vehicle, A vs. vehicle of corresponding vehicle group, B vs. control corresponding Wnt-C59-treated group, C vs. Wnt-C59 + Wnt3a group, D vs. Wnt-C59 + Wnt5a group, E vs. Wnt-C59 + Wnt7a group, F vs. Wnt-C59 + Wnt5b group, G vs. Wnt-C59 + Wnt11 group.

Figure 10.

Wnt signaling regulates macrophage polarization and cell recruitment to rough Ti implants. C57Bl/6 mice were treated with Wnt-C59 or DMSO vehicle for 3d prior to and every other day following implantation. Csf1r-iCre+; Wls^fl/fl experimental mice and Csf1r-iCre-; Wls^fl/fl littermate control mice were injected with tamoxifen for 5d prior to implantation (n=6 per group). Cells were isolated from implant surfaces 7d post-operatively and subjected to flow cytometry. A) Total Macrophages were identified as CD68+, Pro-Inflammatory as CD68+/CD80+, and AntiInflammatory as CD68+/CD206+. B) MSCs were identified as CD11b-/Sca-1+/CD105+, CD4 T-cells as CD3+/CD4+, and CD8 T-cells as CD3+/CD8a+. p<0.05: # vs. Control, $ vs. Vehicle, A vs. Wls^fl/fl.

Figure 11.

Focal adhesion kinase inhibition impairs surface-dependent Wnt upregulation. Macrophages were treated with 5uM PF-573228 and plated on surfaces for 24 hours prior to harvest and qPCR (n=6 per group). A) Heat map summarizing changes in expression of 19 Wnt-encoding genes in control and FAK-inhibited macrophages. B) Comparison of 6 Wnt ligands previously shown to be involved in macrophage surface-mediated polarization. p<0.05: # vs. TCPS, $ vs. Smooth, % vs. Rough, A vs. Control of corresponding group.