Three-Dimensionally Cultured Jaw Periosteal Cells Attenuate Macrophage Activation of CD4+ T Cells and Inhibit Osteoclastogenesis

Abstract

The implementation of a successful therapeutic approach that includes tissue-engineered grafts requires detailed analyses of graft-immune cell interactions in order to predict possible immune reactions after implantation. The phenotypic plasticity of macrophages plays a central role in immune cell chemotaxis, inflammatory regulation and bone regeneration. The present study addresses effects emanating from JPC-seeded β-TCP constructs (3DJPCs) co-cultivated with THP-1 derived M1/M2 macrophages within a horizontal co-culture system. After five days of co-culture, macrophage phenotype and chemokine secretion were analyzed by flow cytometry, quantitative PCR and proteome arrays. The results showed that pro-inflammatory factors in M1 macrophages were inhibited by 3DJPCs, while anti-inflammatory factors were activated, possibly affected by the multiple chemokines secreted by 3D-cultured JPCs. In addition, osteoclast markers of polarized macrophages were inhibited by osteogenically induced 3DJPCs. Functional assays revealed a significantly lower percentage of proliferating CD4⁺ T cells in the groups treated with secretomes from M1/M2 macrophages previously co-cultured with 3DJPCs compared to controls without secretomes. Quantifications of pit area resorption assays showed evidence that supernatants from 3DJPCs co-cultured with M1/M2 macrophages were able to completely suppress osteoclast maturation, compared to the control group without secretomes. These findings demonstrate the ability of 3D cultured JPCs to modulate macrophage plasticity.

Keywords: CD4+ T cells; THP-1; jaw periosteal cells; macrophage polarization; mesenchymal stem cells; osteoclastogenesis; pit area resorption assay; secretomes; β-tricalcium phosphate scaffolds.

Figure 1

CD80 and CD86 surface marker expression of M1-macrophage co-cultures. Representative histograms (light yellow peaks: isotype control; red peaks: target markers) and graphs showing the CD80 (a) and CD86 (b) expression of M1 macrophages after five days of co-culture with 3DJPCs/3DOBJPCs, as detected by flow cytometry. Data are displayed as means ± SEM and analyzed by one-way ANOVA (n = 3, * = p < 0.05, ** = p < 0.01), ns = not significantly different.

Figure 2

CD86 and CD209 surface marker expression of M2-macrophage co-cultures. Representative histograms (light yellow peaks: isotype control; red peaks: target markers) and graphs showing the percentage of the CD86 (a) and CD209 (b) positivity of M2 macrophages after five days of co-culture with 3DJPCs/3DOBJPCs, as detected by flow cytometry. Data are displayed as means ± SEM and analyzed by one-way ANOVA (n = 3, * = p < 0.05, ** = p < 0.01, **** = p < 0.0001), ns = not significantly different.

Figure 3

Gene expression of TNF-α, CCL-2, CCL3, CXCL10, IL-10, CD163, CD209, VEGFA, and TGF-β₁ in M1 macrophages co-cultured for 5 days with 3DJPCs/OB3DJPCs (3DJPCs/OB3DJPCs-M1 groups) compared to M1 macrophages co-cultured with cell-free β-TCP scaffolds (TCP/OBTCP-M1 groups). Relative fold gene expression was calculated using the 2^{−(∆∆Ct)} method and mRNA levels in the TCP-M1 group were set as 1. Data are displayed as means ± SEM and analyzed by one-way ANOVA (n = 3, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001), ns = not significantly different.

Figure 4

Gene expression of TNF-α, CCL-2, CCL3, CXCL10, IL-10, CD163, CD209, VEGFA, and TGF-β₁ in M2 macrophages co-cultured for 5 days with untreated/osteogenically induced 3DJPCs (3DJPCs/3DOBJPCs-M2 groups) and cell-free β-TCP scaffold controls (TCP/OBTCP-M2 groups). Relative fold gene expression was calculated using the 2^{−(∆∆Ct)} method and mRNA levels in TCP-M2 were set to 1. Data are displayed as means ± SEM and analyzed by one-way ANOVA (n = 3, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001), ns = not significantly different.

Figure 5

Expression of osteoclastogenesis-related genes (CTSK, ACP5 and RANK) in M1/M2 macrophages co-cultured for 5 days with untreated/osteogenically induced 3DJPCs or cell-free β-TCP scaffolds. (a): Gene expression in co-cultured M1 macrophages. (b): Gene expression in co-cultured M2 macrophages. Relative fold gene expression was calculated using the 2^{−(∆∆Ct)} method and mRNA levels in TCP-M1 or TCP-M2 groups were set to 1. Data are displayed as means ± SEM and analyzed by one-way ANOVA (n = 3, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001), ns = not significantly different.

Figure 6

RUNX2, ALPL, OCN, RANKL, OPG, G-CSF, HLA-DR, CSF-1, IL-6 and CXCL12 gene expression of 3DJPCs/3DOBJPCs co-cultured for 5 days with M1/M2 macrophages analyzed by quantitative PCR. Relative fold gene expression was calculated using the 2^{−(∆∆Ct)} method and mRNA levels in the 3DJPC co-cultured with M1 macrophages (M1-3DJPC group) were set to 1. Data are displayed as means ± SEM and analyzed by one-way ANOVA (n = 3, * = p < 0.05, ** = p < 0.01), ns = not significantly different.

Figure 7

Chemokine secretion in supernatants of M1 macrophages co-cultured with 3DJPCs/3DOBJPCs was analyzed with proteome profiler arrays. (a): X-rays of representative membranes showing chemokine dot blots in different groups (rectangle: detected differential spots between groups). (b): Quantification of pixel densities by the ImageJ software for the chemokine dot blots marked in a. Data are displayed as means ± SEM and analyzed by one-way ANOVA (n = 3, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001), ns = not significantly different.

Figure 8

Chemokine secretion in supernatants of M2 macrophages co-cultured with 3DJPCs/3DOBJPCs was analyzed with proteome profiler arrays. (a): X-rays of representative membranes with chemokine dot blots in different groups (rectangle: detected differential spots between groups). (b): Quantification of pixel densities by the ImageJ software for chemokine dot blots marked in a. Data are displayed as means ± SEM and analyzed by one-way ANOVA (n = 3, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001), ns = not significantly different.

Figure 9

Effects of M1/M2 secretomes on the CD4⁺ T cell proliferation ability. (a): Purity of CD4⁺ T cells after isolation using the CD4⁺ T cell isolation kit (%). (b): Analysis of CD4⁺ T cell concentration (cells/mL). (c): Mean fluorescence intensity of proliferating CD4⁺ T cells. Data are displayed as means ± SEM and analyzed by one-way ANOVA (n = 3, * = p < 0.05, ** = p < 0.01, *** = p < 0.001).

Figure 10

Effects of secretomes from JPC chambers after co-culturing with M1/M2 macrophages on osteoclast differentiation of PBMCs. PBMCs were seeded on CaP coated plates and incubated with JPC-secretomes from different M1/M2 co-culture conditions for 6 days. (a): CaP coating was stained by AgNO₃ to visualize osteoclastic resorption pits. (b): Quantification of pit area was performed using the ImageJ software. Data are displayed as means ± SEM and analyzed by one-way ANOVA (n = 3, **** = p < 0.0001). (c): Representative image of PBMCs after 6 days of osteoclast differentiation (positive control). The cells were stained for actin and nuclei by phalloidin (red fluorescence) and Hoechst (blue fluorescence) to detect multinucleated osteoclasts with actin rings. CaP coating was stained by calcein (green fluorescence), and resorption pits are visible as black areas. Scale bar = 500 µm.

Figure 11

Summary of the results obtained in the present study. An overview of activating/inhibiting interactions between 3DJPCs and macrophages including the involved factors is provided. This illustration was created with BioRender.com, accessed on 20 October 2022.

Figure 12

The co-culture procedure and the examined groups. (a): experimental flow chart of pre-culture and co-culture of 3DJPCs/3DOBJPCs and M1/M2 macrophage groups. (b): experimental setup of co-culture and control groups.