CD301b+ macrophage: the new booster for activating bone regeneration in periodontitis treatment

Abstract

Periodontal bone regeneration is a major challenge in the treatment of periodontitis. Currently the main obstacle is the difficulty of restoring the regenerative vitality of periodontal osteoblast lineages suppressed by inflammation, via conventional treatment. CD301b⁺ macrophages were recently identified as a subpopulation that is characteristic of a regenerative environment, but their role in periodontal bone repair has not been reported. The current study indicates that CD301b⁺ macrophages may be a constituent component of periodontal bone repair, and that they are devoted to bone formation in the resolving phase of periodontitis. Transcriptome sequencing suggested that CD301b⁺ macrophages could positively regulate osteogenesis-related processes. In vitro, CD301b⁺ macrophages could be induced by interleukin 4 (IL-4) unless proinflammatory cytokines such as interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) were present. Mechanistically, CD301b⁺ macrophages promoted osteoblast differentiation via insulin-like growth factor 1 (IGF-1)/thymoma viral proto-oncogene 1 (Akt)/mammalian target of rapamycin (mTOR) signaling. An osteogenic inducible nano-capsule (OINC) consisting of a gold nanocage loaded with IL-4 as the "core" and mouse neutrophil membrane as the "shell" was designed. When injected into periodontal tissue, OINCs first absorbed proinflammatory cytokines in inflamed periodontal tissue, then released IL-4 controlled by far-red irradiation. These events collectively promoted CD301b⁺ macrophage enrichment, which further boosted periodontal bone regeneration. The current study highlights the osteoinductive role of CD301b⁺ macrophages, and suggests a CD301b⁺ macrophage-targeted induction strategy based on biomimetic nano-capsules for improved therapeutic efficacy, which may also provide a potential therapeutic target and strategy for other inflammatory bone diseases.

Fig. 1

Periodontal bone exhibits a degree of repair after periodontal inflammation subsides. a Schematic illustration of a murine periodontitis model establishment in the inflammatory and healing stages with ligature persistence and removal. M1–3, the three molars. b Bone level changes on the buccal side during the inflammatory and healing phases of periodontitis were calculated relative to the non-ligated (NL) contralateral site (baseline) (n = 4). c Representative images of immunofluorescent staining for ALP and TRAP on periodontal tissue sections from the 8DL and 10DR groups. D dentin, B bone. Scale bar, 100 μm. d Quantification of ALP-positive area and TRAP-positive osteoclast number on sagittal sections from the 8DL and 10DR groups (n = 4). e Alterations in mRNA expression of the inflammatory cytokines IL-1β and TNF-α in periodontal lesions from different groups. Data were normalized to GADPH mRNA and are presented as fold change relative to baseline, set as 1 (n = 4). f Representative immunohistochemistry micrographs of 8DL and 10DR groups identifying IL-1β and TNF-α. Arrows indicate positive cells in the slide. Scale bar, 100 μm

Fig. 2

CD301b⁺ macrophages are enriched at the frontier of periodontal bone repair. a Representative flow cytometry contour plots of CD11b⁺F4/80⁺CD301b⁺ macrophages (Φ) throughout the progression and healing stages of periodontitis. b Quantification of the percentage of CD301b⁺ macrophages within periodontal lesions at indicated timepoints (n = 3). c Correlations between the percentages of CD301b⁺ and CD206⁺ macrophages at different timepoints. d Immunofluorescent staining of periodontal tissue sections from the 6DR group to identify the locations of CD301b⁺CD206⁺, CD301b⁺CD206^-, and CD301b^-CD206⁺ macrophages. The yellow frame indicates the locally amplified area. D dentin, B bone. Scale bar, 100 μm

Fig. 3

CD301b⁺ macrophages are required for periodontal bone repair. a The study protocol of periodontitis model establishment in Mgl2^DTR mice injected with PBS or DT to investigate periodontal bone changes during the resolving phase. b Representative images of μCT analysis and H&E staining to observe bone loss (bidirectional red arrows) in the DT and PBS group. Scale bar, 500 μm. c Based on three-dimensional μCT reconstruction, bone loss and other relevant bone parameters (BV/TV, Tb.Th, Tb.Sp) are quantified (n = 4). d Immunofluorescence staining of the osteogenic marker OSX and ALP in periodontal tissue sections from CD301b⁺ macrophage-depleted and non-depleted groups followed by corresponding H&E staining. D dentin, B bone. Scale bar, 100 μm. e Quantitative analysis of the proportion of OSX⁺ cells and the proportion of ALP⁺ areas in two groups (n = 3)

Fig. 4

CD301b⁺ macrophage-derived IGF-1 promotes osteogenic differentiation via the IGFR/AKT/mTOR pathway. a Study design illustrating the sorting of CD301b⁺ macrophages from periodontal tissue in vivo for RNA-Seq assays, and from IL-4-induced BMDMs in vitro for cellular experiments. b Heatmap of the top 78 upregulated genes associated with regulation of osteogenesis in the GO analysis. c Flow cytometry analysis and quantification of CD301b⁺ macrophages with or without IL-4 induction of BMDMs for 24 h (n = 3). d Cellular immunofluorescence for CD301b in IL-4-activated BMDMs incubated with or without IL-1β and TNFα for 24 h. The number of CD301b⁺ macrophages per field was quantified (n = 3). e, f ALP and alizarin red staining to reveal the effects of CM collected from different groups on the osteogenic induction of BMSCs for 7 and 14 days. g Western blotting detection of p-IGFR, IGFR, p-Akt, Akt, p-mTOR, and mTOR in BMSCs with different treatments

Fig. 5

Design, characterization, and functional verification of OINCs. a Pattern diagram of the design and synthesis of OINCs. b Representative transmission electron microscopy image of AuNC and OINC. Scale bar, 50 nm. c Dynamic light scattering measurements indicating hydrodynamic size and zeta potential of AuNCs and OINCs. d Ultra-violet-visible absorption spectra of AuNCs and OINCs under different wavelengths of irradiation. e Release rate curve of OINCs with or without far-red irradiation. f, g Representative photothermal images of OINCs injected into periodontal tissue under 690-nm far-red irradiation, and temperature-time curves of AuNCs and OINCs compared with the normal saline group. h Characteristic protein bands of AuNCs, neutrophil lysates, and OINCs resolved by western blotting. i Binding capacity of different concentrations of OINCs with IL-1β and TNF-α

Fig. 6

OINCs promote periodontal bone regeneration by inducing the formation of CD301b⁺ macrophages. a Flowchart of the time-course of OINC administration and irradiation in a mouse periodontitis model. b Representative images of immunofluorescent staining for CD301b⁺ macrophages and ALP, and corresponding H&E staining in the different groups including normal saline (NS), AuNCs, and OINCs. c Quantitative analysis of CD301b⁺ macrophage number and ALP⁺ area per slide (n = 3). d μCT analysis and Masson staining to compare levels of regenerative bone in different groups. e Quantification of bone-associated parameters (bone loss, BV/TV) to evaluate the therapeutic efficacy of OINCs (n = 4)