Vitamin D Receptor Genetic Polymorphisms Associate With a Decreased Susceptibility to Extremity Osteomyelitis Partly by Inhibiting Macrophage Apoptosis Through Inhibition of Excessive ROS Production via VDR-Bmi1 Signaling

Abstract

Background: Previous studies had reported that vitamin D receptor (VDR) gene polymorphisms were related to the development of several inflammatory disorders. However, potential links between such variations and the risk of developing a bone infection and underlying mechanisms remain unclear. This study aimed to analyze potential associations between VDR genetic variations and susceptibility to extremity osteomyelitis (OM) in a Chinese Han population and investigate potential mechanisms. Methods: Between January 2016 and August 2020, altogether 398 OM patients and 368 healthy controls were genotyped for six VDR gene polymorphisms, including ApaI (rs7975232), BsmI (rs1544410), FokI (rs2228570), TaqI (rs731236), GATA (rs4516035), and Cdx-2 (rs11568820) by the SNaPshot genotyping method. Then, male C57BL/6 mice were randomly divided into vitamin D-standard, -excess, -deficient, and -rescued groups. One week after making the model surgery, OM occurrence and severity were assessed using the bacterial count and histopathological staining. In vitro, phagocytosis, apoptosis, and bactericidal ability of macrophages were evaluated by overexpression or knockdown of VDR protein. Results: Significant associations were found among rs7975232, rs1544410, and OM development by the recessive model (AA vs. AC + CC, p = 0.037, OR = 0.594), homozygous model (AA vs. CC, p = 0.033, OR = 0.575), and heterozygous model (CT vs. CC, p = 0.049, OR = 0.610), respectively. Patients with the AA genotype of rs7975232 had a relatively higher mean level of vitamin D than those with AC and CC genotypes (22.5 vs. 20.7 vs. 19.0 ng/ml). Similarly, patients with CT genotype of rs1544410 had a relatively higher mean vitamin D level than those with CC genotype (20.94 vs. 19.89 ng/ml). Outcomes of in vivo experiments showed that the femoral bacterial load of vitamin D-deficient mice was highest among different vitamin D dose groups, with the most severe histopathological features of infection, and vitamin D supplementation partly reversed the changes. While in vitro experiment results revealed that active vitamin D promoted phagocytosis and sterilization of macrophages and inhibited apoptosis during infection. Reactive oxygen species (ROS) inhibitor inhibited apoptosis of macrophages induced by bacterial infection. Active vitamin D inhibited excessive ROS production in macrophages via the VDR-Bmi1 signaling pathway. Conclusion: In this Chinese cohort, ApaI and BsmI are associated with a decreased risk of OM development by influencing serological vitamin D level, the latter of which reduced macrophage apoptosis with inhibition of excessive ROS production via the VDR-Bmi1 signaling pathway.

Keywords: ROS–reactive oxygen species; genetic polymorphism; macrophages; osteomyelitis (OM); vitamin D receptor (VDR).

FIGURE 1

Preoperative serum levels of vitamin D among different genotypes of rs7975232 (A), rs1544410 (B), and rs2228570 (C) in the OM patients. *p < 0.05, **p < 0.01, and ***p < 0.001.

FIGURE 2

Vitamin D supplementation inhibited the risks of IAI occurrence and severity. (A) After the femoral implant was removed, the infected femur was ground and made into a homogenized solution. Representative images after coating the plate with the homogenized solution overnight. (B) CFUs of homogenized solution in each group. (C) Representative images of H&E staining. Magnification, ×40. (D) Smeltzer’s score. Mean value of two experienced reviewers’ scores. (E) Double immunofluorescence staining for S. aureus (red) and F4/80⁺ macrophages (green). Magnification, ×200. (F) Count of S. aureus in view. (G) Count of F4/80⁺ macrophages in view.

FIGURE 3

Vitamin D enhanced macrophage phagocytosis and bactericidal ability. (A) Active vitamin D gradient promoted VDR protein expression. (B) siRNAs inhibited the expression of VDR protein, and the inhibitory effect of siVDR3 was the most obvious. (C) Representative images after coating the plate with overexpression or knockdown of VDR protein levels. (D) CFUs of 1 hour after bacterial infection. (E) Double immunofluorescence staining for S. aureus (red) and F-Actin (cytoskeleton, green). Magnification, ×2,000. (F) Count of S. aureus intracellular or extracellular. “ns” for not significant, *p < 0.05, **p < 0.01, and ***p < 0.001.

FIGURE 4

Vitamin D inhibited macrophage apoptosis induced by S. aureus associated infection. (A) Apoptosis detection, Caspase-3/7 (green) and PI (red). Magnification, ×200. (B) Result of western blotting for apoptosis-related proteins. “ns” for not significant, *p < 0.05, **p < 0.01, and ***p < 0.001.

FIGURE 5

Vitamin D inhibited excessive ROS expression during S. aureus associated infection. (A) ROS generation fluorescence detection, ROS (green). Magnification, 1,000×. (B) Mean grey value of ROS generation. (C) Apoptosis detection, caspase-3/7 (green) and PI (red). Magnification, ×200. (D) Result of western blotting for apoptosis-related proteins. “ns” for not significant, *p < 0.05, **p < 0.01, and ***p < 0.001.

FIGURE 6

Inhibition of Bmi1 weakened the protective effect of vitamin D on macrophages. (A) Result of western blotting for apoptosis-related proteins. (B) Apoptosis detection, caspase-3/7 (green) and PI (red). Magnification, ×400. “ns” for not significant, *p < 0.05, **p < 0.01, and ***p < 0.001.