Potential diagnostic value of PD‐1 in peripheral blood mononuclear cells of postmenopausal osteoporosis patients

Abstract Background Postmenopausal osteoporosis (PMOP) is an estrogen deficiency‐induced skeletal disorder. Bone mineral density (BMD) testing is the gold standard for diagnosing osteoporosis. However, its sensitivity for fracture risk assessment is low. Programmed cell death protein 1 (PD‐1) is a key immune checkpoint molecule implicated in the pathophysiology of bone remodeling, but its role in osteoporosis has not yet been explored. Thus, this study aimed to assess the expression and diagnostic utility of PD‐1 in PMOP. Methods A total of 56 patients with PMOP and 37 postmenopausal healthy controls (NC) were enrolled in the study. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation, and PD‐1 expression was measured by quantitative polymerase chain reaction (qPCR). Pearson's correlation test was performed to explore the associations between PD‐1 level and clinical variables, while receiver operating characteristic (ROC) curve analysis was used to evaluate the potential diagnostic value of PD‐1 in patients with PMOP. Results We found that PD‐1 level was significantly upregulated in the PBMCs of PMOP patients than those of NC (P = .016). PD‐1 expression was positively correlated with C‐reactive protein (CRP) levels. ROC curve analysis showed that PD‐1 had certain diagnostic value for PMOP (area under the curve = 0.65, standard error = 0.06, 95% confidence interval [0.53,0.76], P = .016), with a sensitivity and specificity of 44.64% and 81.08%, respectively. Conclusion Programmed cell death protein 1 is significantly upregulated in the PBMCs of PMOP patients and has certain diagnostic value for PMOP.


| INTRODUC TI ON
Postmenopausal osteoporosis (PMOP) is a systemic, metabolic bone disorder resulting from decreased ovarian estrogen production after menopause 1 and disrupted balance between bone resorption (mediated by osteoclasts) and bone formation (mediated by osteoblasts). Estrogen deficiency can manifest as increased bone resorption 2 and osteoporotic fractures, which can seriously affect the quality of life of middle-aged and elderly women. Bone mineral density (BMD) testing using dual-energy X-ray absorptiometry (DXA) is the gold standard for diagnosing osteoporosis. However, the technique is limited because changes to bone density occur slowly and can only be measured meaningfully over the course of several years. Measurement of bone turnover markers (BTMs) is useful in differential diagnosis, distinguishing the different types of osteoporosis, and early assessment of patient response to treatment, but it cannot be used to diagnose osteoporosis. Therefore, there is an urgent need to identify more sensitive diagnostic markers for osteoporosis.
The recent advances in osteoimmunology, an interdisciplinary research field that explores the interaction between bone and immune system, revealed that bone remodeling is under strict immunological control. 3 Thus, it is of vital clinical importance to identify the specific immune molecules involved, as they could serve as potential targets for the diagnosis and treatment of osteoporosis.
Programmed cell death protein-1 (PD-1) is an inhibitory immune receptor that is mainly expressed on active T cells, B cells, monocytes, dendritic cells, and natural killer cells. PD-1 has two ligands, of which programmed death ligand-1 (PD-L1) is considered to be the primary ligand. The PD-1/PD-L1 pathway plays a critical role in the pathophysiology of autoimmune diseases, tumorigenesis, chronic infections, and inflammation. 4 A recent study found that activation of the PD-1/PD-L1 pathway was associated with hyper-activation of osteoclasts and impaired T-cell proliferation in multiple myeloma. 5 However, its role in osteoporosis has not yet been studied. To the best of our knowledge, no clinical studies to date have investigated PD-1 levels in peripheral blood mononuclear cells (PBMCs) of osteoporosis patients. Therefore, we explored PD-1 expression, and its association with clinical and laboratory parameters, in PMOP patients.  T-score at the lumbar spine or femur ≤ −2.5 standard deviations (SD). and (d) patients with cancer or severe heart, blood, or mental disorders.

| Isolation of PBMCs
A sample of venous blood was collected from all participants. The PBMCs were isolated by density gradient separation at 18-20°C using Ficoll-Paque PLUS (GE Healthcare), according to the manufacturer's instructions. The PBMCs were isolated within 6 hours of blood collection to optimize their viability.

| Total RNA extraction
Total RNA was extracted from the isolated PBMCs using TRIzol reagent (Invitrogen, Karlsruhe, Germany) according to the manufacturer's instructions.

| Quantitative PCR (qPCR)
Polymerase chain reaction was performed on the Rotor-Gene Q qPCR cycler (QIAGEN), using the UltraSYBR Mixture (High ROX) (CWBiotech). The PCR primers were designed and synthesized by Sangon Biotech Co., Ltd. (Table 1). The relative expression of PD-1 was normalized to that of human β-actin, using the 2 −ΔΔCT method.

| Statistical analyses
All statistical analyses were performed using GraphPad Prism version 6.0. Continuous variables were expressed as mean ± SD and compared between the groups using unpaired t test. Differential PD-1 expression was analyzed by t test, and the results were presented as a scatter plot. Linear associations between parameters were evaluated using Pearson's correlation. The diagnostic value of PD-1 was assessed by receiver operating characteristic (ROC) curve analysis, in which the PMOP patients and healthy controls served as true positives and true negatives, respectively. The difference was considered statistically significant when P < .05.

| Characteristics of study subjects
Participant age, white blood cell count (WBC), C-reactive protein (CRP), lymphocyte count (LYM), and erythrocyte sedimentation rate (ESR) of the menopausal women did not differ significantly between the PMOP and control groups ( Table 2).

| Expression of bone turnover markers (BTMs)
The T-score at the lumbar spine or femur, and expression of bone turnover markers (BTMs), differed significantly between the two groups. The levels of procollagen type 1 N-terminal propeptide (PINP), osteocalcin (OSTEOC), and β-crosslaps (CROSSL) were higher in patients with PMOP than in controls (Table 3).

| PD-1 expression
We performed qPCR to measure the expression levels of PD-1 in the PMOP and control groups. PD-1 was significantly upregulated in the PMOP group compared with the control group (P = .016) ( Figure 1A).

| Correlation between PD-1 expression and clinical variables
To determine whether PD-1 in the PBMCs of PMOP patients could serve as a biomarker for the severity of PMOP, we evaluated the correlations between PMOP-related clinical features and PD-1 expression in PMOP patients. PD-1 level was correlated with CRP level (r = .334, P < .05), but not with patient age, WBC count, LYM, ESR, BMD, T-score, or CROSSL, PINP, or OSTEOC expression (Table 4).

| ROC curve analysis
We performed ROC curve analysis to assess the diagnostic value of  (Table 5).

| D ISCUSS I ON
Postmenopausal osteoporosis is a common bone disease characterized by reduced bone mass, microstructural destruction of bone tissue, increased bone fragility, and high risk of fracture. It usually    There are several limitations to this study. Firstly, the number of patients was relatively small and derived from a single center.
Larger studies in different racial and regional groups are warranted.
Secondly, diagnostic utility of PD-1 to distinguish PMOP from senile osteoporosis, secondary osteoporosis, and other orthopedic diseases was not evaluated. Finally, the role of PD-1 in PMOP was not explored in vivo. Our future studies will endeavor to address these shortcomings.
To the best of our knowledge, we are the first to determine the expression of PD-1 in the PBMCs of PMOP patients. Our findings help to explain the role of PD-1 in the pathogenesis and progression of the disease, and suggest that measurement of PD-1 levels in the PBMCs has potential diagnostic application for PMOP. Future studies should explore the molecular mechanisms underlying the role of PD-1 in PMOP.

AUTH O R CO NTR I B UTI O N S
X-P C involved in total RNA extraction, reverse transcription, quantitative PCR, statistical analysis, and paper writing; Q Z performed PBMC isolation and clinical data collection; Z-D G performed PBMC isolation; S-J L involved in total RNA extraction and reverse transcription; Z-X C involved in patient enrollment; M-Y C carried out BMD measurement; L Z is the project supervisor; K-W Z is the project leader. Abbreviations: AUC, area under the ROC curve; BTMs, bone turnover markers; CI, confidence interval; CROSSL, β-crosslaps; OSTEOC, osteocalcin; PD-1, programmed cell death protein 1; PINP, procollagen type 1 N-terminal propeptide.