Transient Receptor Potential Vanilloid 4 Is Required for Foreign Body Response and Giant Cell Formation

Abstract

The presence of biomaterials and devices implanted into soft tissue is associated with development of a foreign body response (FBR), a chronic inflammatory condition that can ultimately lead to implant failure, which may cause harm to or death of the patient. Development of FBR includes activation of macrophages at the tissue-implant interface, generation of destructive foreign body giant cells (FBGCs), and generation of fibrous tissue that encapsulates the implant. However, the mechanisms underlying the FBR remain poorly understood, as neither the materials composing the implants nor their chemical properties can explain triggering of the FBR. Herein, we report that genetic ablation of transient receptor potential vanilloid 4 (TRPV4), a Ca²⁺-permeable mechanosensitive cation channel in the transient receptor potential vanilloid family, protects TRPV4 knockout mice from FBR-related events. The mice showed diminished collagen deposition along with reduced macrophage accumulation and FBGC formation compared with wild-type mice in a s.c. implantation model. Analysis of macrophage markers in spleen tissues and peritoneal cavity showed that the TRPV4 deficiency did not impair basal macrophage maturation. Furthermore, genetic deficiency or pharmacologic antagonism of TRPV4 blocked cytokine-induced FBGC formation, which was restored by lentivirus-mediated TRPV4 reintroduction. Taken together, these results suggest an important, previously unknown, role for TRPV4 in FBR.

Figure 1

Transient receptor potential vanilloid 4 (TRPV4) deletion in mice prevents macrophage accumulation, foreign body giant cell (FBGC) formation, and collagen accumulation in a s.c. implantation model. Images of sections of filters (asterisks) implanted subcutaneously for 28 days in wild-type (WT) and TRPV4 knockout (KO) mice are shown. A: Sections were stained with Masson trichrome to show deposition of collagen (blue). Red asterisks indicate tissue-implant interface. B: Quantification of collagen in experiment shown in A. C: FBGCs were stained by hematoxylin and eosin. Black arrows indicate presence of FBGCs in sections of implants from WT mice. Red asterisks indicate tissue-implant interface. D: Quantification of FBGC numbers from experiment shown in C. E: Sections were stained with CD68 IgG and visualized with Alexa Fluor 594–conjugated secondary IgG (red). Nuclei were stained with DAPI (blue). Yellow arrows indicate presence of macrophages at tissue-implant interface. White asterisks indicate tissue-implant interface. F: Quantification of macrophage accumulation in experiment shown in E. n = 5 mice per group (A, C, E). ^∗∗∗P ≤ 0.001 (t-test). Scale bars = 50 μm (A, C, E). HPF, high-power field.

Figure 2

Basal macrophage differentiation is normal in transient receptor potential vanilloid 4 (TRPV4)–deficient mice. A: Sections of spleens from wild-type (WT) and TRPV4 knockout (KO) mice were stained with antibodies for CD68 (FA11), F4/80 (EMRI), 5D3 (mannose receptor), and ED31 (MARCO); nuclei were stained with DAPI. Staining by secondary antibody in the absence of primary antibody was used as IgG control (cont.). B: Quantification of macrophage abundance. C: Percentages of F4/80⁺/CD11b⁺ double-positive cells were assessed by fluorescence-activated cell sorting (FACS) analysis. Cells were collected from the peritoneal cavity [resident peritoneal macrophages (ResMΦ)], the spleen [spleen macrophages (SpleenMΦ)], and the peritoneal space of thioglycolate-injected mice [thioglycolate macrophages (ThioMΦ)]. Data are expressed as means ± SD (B and C). n = 5 mice per group (B); n = 3 animals (C). Scale bars = 50 μm.

Figure 3

Transient receptor potential vanilloid 4 (TRPV4) deficiency results in impaired macrophage fusion. Bone marrow–derived macrophages (BMDMs) from wild-type (WT) and TRPV4 knockout (KO) mice were stimulated with IL-4 + granulocyte-macrophage colony-stimulating factor (GM-CSF) for 6 days to induce macrophage fusion and foreign body giant cell (FBGC) formation. A: Giemsa staining shows the presence of multinucleated FBGCs (arrows). B: Bright-field images. C: DAPI staining of nuclei. D–F: Quantification of data from experiment shown in A–C. D: Number of FBGCs per high-power field (HPF). E: Percentage macrophage fusion. F: Nuclei per FBGC. G: WT and TRPV4 KO BMDMs were labeled with red-fluorescent Qdot-655 or green-fluorescent Qdot-525 nanoparticles, and fusion was induced by stimulation with IL-4 + GM-CSF. Fusion of macrophages is represented by colocalization of the red and green fluorescent labels (yellow). Nuclei were stained with DAPI (blue). H: Quantitation of colocalization. Red, yellow, or white arrows indicate presence of FBGCs. Data are expressed as means ± SEM (D–F and H). n = 3 independent experiments (A–C, G). ^∗∗∗P ≤ 0.001, ^∗∗∗∗P ≤ 0.0001 (t-test). Scale bars = 100 μm (A–C and G).

Figure 4

Transient receptor potential vanilloid 4 (TRPV4) plays a direct role in foreign body giant cell (FBGC) formation. Wild-type (WT) bone marrow–derived macrophages (BMDMs) treated with GSK2193874 (GSK219) or vehicle were stimulated with IL-4 + granulocyte-macrophage colony-stimulating factor (GM-CSF), as described in Figure 3, to induce macrophage fusion. A–C: Quantification of results. A: Number of foreign body giant cells (FBGCs) per high-power field. B: Percentage macrophage fusion. C: Nuclei per FBGC. A t-test was performed. D: To assess the effect of activation of TRPV4 by GSK1016790A (GSK101) on FBGC formation, WT BMDMs were treated with GSK101 or vehicle, and then stimulated with IL-4 + GM-CSF to induce FBGC formation. Giemsa-stained images shown are representative of five different fields per condition. E–H: Quantification of data from experiment shown in D. E: Number of FBGCs per high-power field. F: Percentage macrophage fusion. G: Nuclei per FBGC. H: Average size of FBGCs. A t-test was performed. I: Images are representative of five different fields per condition to assess the effect of TRPV4 gene-expressing constructs containing lentiviral particles [lenti-TRPV4–green fluorescent protein (GFP)] or control (cont.) GFP constructs containing lentiviral particles (lenti-GFP) expression in TRPV4 knockout (KO) BMDMs on FBGC formation. Arrows indicate the presence of FBGCs. J: Quantification of number of FBGCs per high-power field (HPF). Data are expressed as means ± SEM (B, C, E–H, and J). n = 3 independent experiments (B, C, E–H, and J). ^∗P ≤ 0.05, ^∗∗P ≤ 0.01, ^∗∗∗P ≤ 0.001, and ^∗∗∗∗P ≤ 0.0001 (t-test). Scale bars: 100 μm (D); 50 μm (I).