Neglected immunoregulation: M2 polarization of macrophages triggered by low‐dose irradiation plays an important role in bone regeneration

Current studies have found that low‐dose irradiation (IR) can promote bone regeneration. However, mechanism studies of IR‐triggered bone regeneration mainly focus on the effects of osteoblasts, neglecting the role of the surrounding immune microenvironment. Here in this study, in vitro proliferation experiments showed that low‐dose IR ≤2 Gy could promote the proliferation of bone marrow mesenchymal stem cells (BMSCs), and qRT‐PCR assay showed that low‐dose IR ≤2 Gy could exert the M2 polarization of Raw264.7 cells, while IR >2 Gy inhibited BMSC proliferation and triggered M1 polarization in Raw264.7 cells. The ALP and mineralized nodules staining showed that low‐dose IR ≤2 Gy not only promoted osteoblast mineralization through IR‐triggered osteoblast proliferation but also through M2 polarization of Raw264.7 cells, while high‐dose IR >2 Gy had the opposite effect. The co‐incubation of BMSC with low‐dose IR irradiated Raw264.7 cell supernatants increased the mRNA expression of BMP‐2 and Osx. The rat cranial defects model revealed that low‐dose IR ≤2 Gy gradually promoted bone regeneration, while high‐dose IR >2 Gy inhibited bone regeneration. Detection of macrophage polarity in peripheral blood samples showed that low‐dose IR ≤2 Gy increased the expression of CD206 and CD163, but decreased the expression of CD86 and CD80 in macrophages, which indicated M2 polarization of macrophages in vivo, while high‐dose IR had the opposite effect. Our finding innovatively revealed that low‐dose IR ≤2 Gy promotes bone regeneration not only by directly promoting the proliferation of osteoblasts but also by triggering M2 polarization of macrophages, which provided a new perspective for immune mechanism study in the treatment of bone defects with low‐dose IR.


| INTRODUC TI ON
Bone defects are still a medical concern in recent decades. Millions of bone fixation are performed each year. Bone autografts and allografts, or biologically inert metallic devices-based bone fixation, are the gold standard for the treatment for large bone defects. 1,2 With the development of biomaterials, many synthetic and natural biomaterials with good biocompatibility and biodegradability are explored for the application of implanted scaffolds, such as collagen, 3 calcium phosphate, 4 hydroxyapatite 5,6 and silica. 7 There is no denying that these implants also bring some inevitable side effects when they work. Subsequent surgical removal is often needed for metal bone-fixation devices. 8 The use of bone autografts or allografts often carries a risk of disease transmission from the donor material, additional donor site morbidity and limited availability compared to the need. 9 The biodegradable implanted scaffolds may face biological toxicity, bacterial infection and inflammatory responses. 10 In recent years, more and more researchers have found that in addition to implantation, irradiation (IR) has been found to play a role in bone regeneration. Deloch et al. 11 showed a positive impact of 0.1 and 0.5 Gy on bone formation of healthy osteoblasts. Wright et al. 12 reported that 2 Gy of IR could result in local and systemic bone loss in C57BL/6 mice. Chen et al. 13 showed that 0.5 Gy of IR promoted the fracture repair in Sprague Dawley (SD) rats. In the work of Hu et al., 14 x-ray at 2 Gy decreased the mineralization effect of OCT-1 cells after a single IR. Sun et al. 15 showed that a 2 Gy local X-IR in rats led to an inhibition of osteogenic differentiation. In contrast, the same cell line subjected to the same dose exerted only timedependent cell cycle arrest without significant effects on the proliferation and differentiation effect, 16 while another study further suggested that 2 Gy of x-ray not only increased differentiation and mineralization potential of calvarial osteoblasts but also upregulated the expression of many related cytokines, including alkaline phosphatase (ALP), osteopontin (OPN) and osteocalcin (OCN) during the process. 17 Liu et al. 18 reported that 6 Gy of IR promoted ROS generation by mediating upregulation of miR-22, which in turn promoted apoptosis of bone marrow mesenchymal stem cells (BMSCs).
These studies mainly illustrate the effect of IR on osteoblasts (OBs) and osteoclasts (OCs) in bone tissue. In addition to OBs and OCs, the bone microenvironment also includes various types of cells that affect bone remodelling. 19 It was reported that IR affects not only OBs and OCs but also other cells in the bone microenvironment. 20 IR affects bone vasculature, while bone vasculature has a vital effect on bone homeostasis as a transporter of nutrients and oxygen. 21 Studies showed reduced blood flow in irradiated bones in mice and rats after varying doses of IR. 22 IR also affects immune microenvironment in the bone 23 ; however, the detailed osteoimmunological mechanism has not been revealed. The effect of IR on bone regeneration is not only dependent on changes in OBs and OCs biology, but also closely related to changes in the overall bone microenvironment. Since bone destruction or remodelling is a dynamic process, and some studies have also explored the effect of low-dose IR on bone and bone formation-related cells, there are more related studies that are worth exploring.
Macrophages, as a critical component of the innate immune system, have been demonstrated to exert important regulatory roles in bone homeostasis and repair. 24,25 M1-polarized macrophages play an important role in the initiation and development of inflammation, and produce effector molecules such as reactive oxygen species (ROS), inducible nitric oxide synthase (iNOS), and inflammatory cytokines such as IL-1β, IL-6 and TNFα. Due to their inflammatory properties, chronic activation of M1 macrophages leads to tissue damage. On the other hand, M2-polarized macrophages secrete anti-inflammatory cytokines such as IL-4 and IL-10. In vivo, these major isoforms are not strictly formed, but are interchangeable. 26,27 It was reported that bone repair is positively associated with M2 macrophage function, 28 and regulated M2 macrophages showed a preventive effect on bone loss in murine periodontitis models. 29 Recently, regulated M2 macrophage polarization using scaffold implantation or exosome was found to significantly enhance bone formation or inhibit periodontal bone loss in animal models. 30,31 In recent decades, studies have come to a consensus that macrophages subjected to ≤1 Gy of IR treatment were likely to be prone to M2 polarization (anti-inflammatory), while >2 Gy of IR was more prone to enhance M1 polarization (pro-inflammatory) of macrophages. 32,33 Therefore, the impact of low-dose IR on macrophages and the following impact on bone repair, as a long-neglected mode of action, should be considered.
Here in this study, we explored the effect of low-dose IR ≤2 Gy on OB proliferation and macrophage polarity, revealing the effect of macrophage polarization on bone regeneration. Rats with cranial defects were used as the animal model to study the bone regeneration effect of low-dose IR on bone defects. Our study is helpful to understand the detailed osteoimmunological mechanism of bone regeneration induced by low-dose IR and provides guidance for the development of bone defect-related diseases and osteoimmunology.

| Cell proliferation and viability
The MTT assay was employed to determine the cell proliferation of Messenger RNA (mRNA) was extracted using an RNA-quick purification kit. Then, the RNA samples were added with SuperMix, reverse-transcribed into complementary DNA at 50°C for 15 min, 85°C for 5 s. The qRT-PCR assay was conducted within the mixture using AceQ® qPCR SYBR Green Master Mix. Finally, the mRNA expression of macrophage M1 polarization marker genes IL-1β, iNOS, and M2 polarization marker genes BMP2, CD206, were quantified using a real-time PCR detection system (ViiA7, ThermoFisher Scientific). The relative mRNA expression levels of the target genes were normalized using GAPDH, and gene expression was calculated using the 2 −ΔΔCT method. Primers used in this study are displayed in the Table S1.

| Expression of osteogenic genes
Raw264.7 cells were seeded in 6-well plates (5 × 10 4 per-well), and incubated for 48 h. After culturing, the cells were stimulated by LPS (1 μg/mL) for 24 h, rinsed with PBS, replaced with fresh culture medium, then irradiated with various doses (0, 0.5, 1, 2, 4 Gy) of IR, and continued to incubate for 12 h, and the supernatants were collected for later use (called supernatants 1). After suction of supernatant 1, the cells in the plates were added with LPS (1 μg/mL) for 24 h, rinsed with PBS, replaced with fresh culture medium for additional culture of 12 h, and the supernatants were also collected for later use (called supernatants 2).
Six-well plates were added with 0.1% gelatin solution and placed in the incubator for 1 h, then the gelatin solution was removed.
Afterwards, BMSCs were seeded into 6-well plates (1 × 10 4 cells/ well) and cultured in OIM for 24 h, then added with supernatants 1 or supernatants 2 for 7 days. After that, messenger RNA (mRNA) was extracted using an RNA-quick purification kit. Then, the RNA samples were added with SuperMix, reverse-transcribed into complementary DNA at 50°C for 15 min, 85°C for 5 s. The qRT-PCR assay was conducted within the mixture using AceQ® qPCR SYBR Green Master Mix. Finally, the mRNA expression of BMP-2 and Osterix (Osx) were quantified using a real-time PCR detection system as above. The relative mRNA expression levels of the target genes were normalized using GAPDH, and gene expression was calculated using the 2 −ΔΔCT method. Primers used in this study are displayed in the Table S1.

| In vivo osteogenesis evaluation
All animal experiments were approved by the Nanjing Medical University Ethics Committee. Male Sprague Dawley (SD) rats (200-250 g) were obtained from Suzhou Sinocell Technology Ltd.
The rats were conducted to drilling operations to construct cranial defect models. Briefly, the rats were first anaesthetized by intraperitoneal injection of 1% pentobarbital sodium. Subsequently, an incision with the appropriate size was made over the scalp with a scalpel for the exposure of cranium. The periosteum was splitted for the exposure of underlying bone. A trephine drill was used to create a 5-mm-diameter cranial defect on both sides of the midline under continuous sterile saline irrigation. Finally, the incisions were sutured after drilling.
The rats were randomly divided into five groups after the operation (n = 3). The following treatments were conducted the next day: Group I did not receive any therapy, Group II-V received 0.5, 1, 2 and 4 Gy total body IR by the linear accelerator Infinity (Elekta Corporation), respectively. After 4 weeks of feeding, the rats were sacrificed, the craniums of rats were removed and fixed in formalin.

| Immunohistochemical staining
Immunohistochemical staining was performed to evaluate the expression of osteogenesis specific biomarkers and polarizationrelated markers. The immunohistochemistry of runt-related transcription factor 2 (RUNX2) was performed as follows: the fixed cranium samples were decalcified in 5% EDTA-Na2 (pH = 7.3), followed by dehydrating, embedding in paraffin and making paraffin sections. Then the sections were permeated with Triton X-100, and cultured with 5% goat serum for 1 h. After that, the sections were incubated with the primary antibody of RUNX2 (1:100 dilution) for 12 h at 4°C. Finally, the secondary antibody was added and incubated for 2 h. Similarly, the immunohistochemical staining of CD86 and CD163 was a similar procedure. Finally, the samples were observed by a light microscopy.

| Macrophage polarity in the blood
According to the above methodology, SD rats were modelled with cranial defect, and divided into the following five groups (n = 5) for IR on the second day: Group I served as blank controls without irradiation treatment, Group II-V received 0.5, 1, 2 and 4 Gy IR, respectively. The day of IR was recorded as Day 0, and blood samples were After the labelling of cell surface antibody, the samples were fixed with 4% paraformaldehyde, permeated with intracellular staining perm wash buffer (1X) and stained with PE-anti rat CD86 antibody.
The sampling and detection process of the polarity changes of peripheral blood monocytes in healthy rats after IR were provided by supporting information.

| Statistical analysis
All data are presented as the mean value ± standard deviation from at least three independent measurements. Comparisons among multiple groups were analysed using the one-way anova with Tukey's multiple comparisons test. The statistical comparisons were performed using GraphPad Prism 8 (version 7; GraphPad Software, Inc.). Mean differences with p < 0.05 were considered statistically significant. The corresponding markers in the figures are defined as *p < 0.05, **p < 0.01 and ***p < 0.001, respectively.

| Low-dose IR promotes the proliferation of BMSCs
Previous reports have all agreed that IR can influence the proliferation of osteoblasts, but the reported dose and final effects (positive or negative) remained controversial. [34][35][36] To study the impact of IR doses on the proliferation of BMSCs, the cell viability study and live/ dead staining were conducted. After being treated with different doses (0-4 Gy) of IR, BMSCs were allowed to grow for another 24, 48 or 72 h, and the cell viability were monitored using the MTT assay.
As shown in Figure 1A, at 24 h post-IR, there was almost no difference in cell viability among low-dose IR ≤2 Gy groups, when the IR dose continued to increase to 4 Gy, the relative cell proliferation rate decreased to 0.88-fold of that of the 2 Gy group. At 48 h, the cell proliferation rate of the 4 Gy group was significantly inhibited, which reduced to 0.94-fold of that of the 0 Gy group and 0.91-fold of that of the 2 Gy group. It was noted that extended incubation time to 72 h showed more significant differences. In particular, BMSCs subjected to 2 Gy IR showed the highest BMSC proliferation profile and had a significant difference compared to the 0 Gy control group, the relative cell proliferation rate of which increased to 1.13-fold of that of the 0 Gy group. While 4 Gy group exhibited more significant differences compared with other groups, which had an obvious decline in cells. The effect of IR on the proliferation of macrophages by the MTT assay was also conducted. As shown in Figure S1, there was no significant differences in the proliferation of macrophages between various doses of IR.
In the live/dead staining assay performed at 72 h post-incubation, as shown in Figure 1B

| Low-dose of IR promotes M2 polarization of macrophages
As previous studies revealed that macrophages reside during all stages of fracture repair, which also contribute significantly to determine the destiny of repair process. 37,38 M2 polarization of macrophages could improve the repair outcome with reduced recuperation time. 39,40 Some studies have reported that macrophages subjected to ≤1 Gy of IR treatment were likely to become M2 polarization (anti-inflammatory), while IR >2 Gy was more prone to enhance M1 polarization (pro-inflammatory) of macrophages. 32,33 Therefore, there is a high potential that low-dose IR can indirectly navigate the bone repair process by affecting macrophages po-

| Low-dose IR-induced osteoblast proliferation and macrophage M2-polarization promote osteogenesis
Based on the above results, we concluded that IR ≤2 Gy can pro-   Figure S3) showed that when the IR dose was gradually increased to 2 Gy, the contents of nitric oxide and IL-1 were gradually decreased but the content of IL-10 was gradually increased. However, when the IR dose increased to 4Gy, the contents of nitric oxide and IL-1 increased significantly, and the content of IL-10 decreased significantly. This proves that the changes in the relevant cytokines secreted by macrophages induced by IR mediate the osteogenic changes. F I G U R E 2 Raw264.7 cells were exposed to LPS inflammatory stimulation, then irradiated by different doses of IR, and cultured for 24 h. RT-PCR was used to determine the intracellular mRNA expression of M1 markers (A) IL-1β, (B) iNOS and M2 markers (C) BMP2, (D) CD206. NS: no significant difference, *p < 0.05, **p < 0.01, ***p < 0.001. The error bars in the graphs represent the standard deviation (n = 3).

| Low-dose IR promotes bone regeneration in rat cranial defect model
To reveal the effect of various doses of IR on bone regeneration, in our study, male SD rats with established cranial defects model were employed and subjected to 0.5-4 Gy of IR once. At 28 days post IR, micro-CT was employed to analyse the formation of new bones within the defect region. As shown in Figure 6, the 3D-reconstruction of the cranium in different groups ( Figure 6A

| Low-dose IR promotes M2 polarization of peripheral blood monocytes in vivo
In order to further verify the effect of IR on the polarization of peripheral blood monocytes in cranial defect rats, the orbital blood of rats after various doses of IR (0-4 Gy) at a predetermined time (Day 1,8,14) were collected. Then PBMC was isolated and four M1/M2 antibodies (CD80/CD86 for M1-polarized monocytes and CD206/CD163 for M2-polarized monocytes) were used as the marks of monocytes polarity, and the samples were detected by flow cytometer. It showed on Day 1 ( Figure 8A), for the low-dose IR ≤2 Gy groups, with the increase of IR dose, the proportion of CD80 + CD86 + monocytes decreased, and that of CD163 + CD206 + monocytes increased. In the 0 Gy control group ( Figure 8B, Day 1), the proportion of CD80 + CD86 + and CD163 + CD206 + monocytes were 32.7% and 6.2%, respectively, while in the 2 Gy group, the proportion of CD80 + CD86 + monocytes reduced to 12.8%, and the proportion of CD163 + CD206 + monocytes increased to 10.3%. When the IR dose comes to 4 Gy, the proportion of CD80 + CD86 + monocytes instead increased to 34.07%, and the proportion of CD163 + CD206 + monocytes instead decreased to 4.72%. On Day 8, in the 0 Gy group ( Figure 8B, Day 8), the proportion of CD80 + CD86 + and CD163 + CD206 + monocytes were 38.3% and 5.6%, respectively, while in the 2 Gy group, the proportion F I G U R E 5 Expression of BMP-2 and Osx in BMSCs after different treatments. The expression of BMP-2 (A) and Osx (B) in BMSCs co-cultured with the supernatants of Raw264.7 cells irradiated with different doses of IR. The expression of BMP-2 (C) and Osx (D) in BMSCs co-cultured with the supernatants of Raw264.7 cells, in which the Raw264.7 cells were irradiated with different doses of IR and then subjected to LPS stimulation. NS: no significant difference, *p < 0.05, **p < 0.01. The error bars in the graphs represent the standard deviation (n = 3).
of CD80 + CD86 + monocytes reduced to 18.8%, and the proportion of CD163 + CD206 + monocytes increased to 17.3%. For the 4 Gy group, the proportion of CD80 + CD86 + monocytes instead increased to 39.4%, and the proportion of CD163 + CD206 + monocytes instead decreased to 6.6%. Day 14 showed a similar trend.
On Day 14, for the low-dose IR ≤2 Gy groups, when the IR of 0 Gy gradually increased to 0.5, 1, 2 Gy, the proportion of CD80 + CD86 + monocytes gradually decreased from 25.9% to 13.4%, while the proportion of CD163 + CD206 + monocytes gradually increased from 1.3% to 17.4%. When the IR dose continued to increase to 4 Gy, the proportion of CD80 + CD86 + monocytes increased to 32.3%, and the proportion of CD163 + CD206 + monocytes decreased to 7.5%. These results indicate that low-dose IR ≤2 Gy can increase the ratio of M2-polarized monocytes and conversely decrease that of M1-polarized ones in peripheral blood. In contrast, from 2 to 4 Gy, IR reduced the ratio of M2-polarized monocytes but increased that of M1-polarized ones in peripheral blood, which exhibited the opposite result.
The effect of IR on the polarization of monocytes in healthy rats was also studied ( Figure S4). Healthy rats showed a similar polarization trend as that in cranial defect rats on Days 1 and 3. Interestingly, the irradiated healthy rats showed faster recovery of the changed polarization state ( Figure S4) as compared with the cranial defect model. It was shown that the polarization differences among different groups were started to unify in healthy rats at Day 14, and there were almost no differences at Day 28. This may be due to the fact that healthy body/organ has a better ability to adjust body indicators to a normal state, which deserves our future exploration.

| CON CLUS IONS
In this study, we found that low-dose IR ≤2 Gy showed enhanced proliferation of BMSCs, while IR >2 Gy showed impaired cell viability. Low-dose IR ≤2 Gy induced M2 polarization of macrophages, while IR >2 Gy was more prone to enhance M1 polarization of macrophages both in vitro and in vivo experiments. Low-dose IR ≤2 Gy promotes bone regeneration not only by directly promoting the proliferation of osteoblasts, but also by triggering M2 polarization of macrophages to promote bone regeneration. The immunologic mechanism and application of low-dose IR on bone regeneration still needs further study and discussion. Tb.Th (D). NS: no significant difference, *p < 0.05, **p < 0.01, ***p < 0.001. The error bars in the graphs represent the standard deviation (n = 3).

F I G U R E 7
Immunohistochemical staining and semiquantitative analysis by imageJ software of RUNX2 (A), CD86 (B) and CD163 (C) in cranium after different doses of IR for 28 days. Brown precipitation represents positive staining. Scale bars, 50 μm. NS: no significant difference, *p < 0.05, **p < 0.01, ***p < 0.001. The error bars in the graphs represent the standard deviation (n = 3).

F I G U R E 8
Flow cytometric results (A) of peripheral blood monocytes M1 (CD80, CD86) and M2 (CD163, CD206) markers from the blood samples of the cranial defect rats and corresponding quantitative results (B) on Days 1, 8 and 14 after IR. NS: no significant difference, *p < 0.05, **p < 0.01, ***p < 0.001. The error bars in the graphs represent the standard deviation (n = 5).