膝骨关节炎患者全血白细胞线粒体DNA复制数量和血浆炎性细胞因子的相关性研究

Relationships between blood leukocyte mitochondrial DNA copy number and inflammatory cytokines in knee osteoarthritis 膝骨关节炎患者全血白细胞线粒体DNA复制数量和血浆炎性细胞因子的相关性研究 1 2 first-author 3 3 3 4 2 3 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:sittisak.h@chula.ac.th †E-mail:sittisak.h@chula.ac.th sittisak.h@chula.ac.th Joint PhD Program in Biomedical Sciences and Biotechnology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand Joint PhD Program in Biomedical Sciences and Biotechnology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand Department of Biochemistry, Osteoarthritis and Musculoskeleton Research Unit, Faculty of Medicine, King Chulalongkorn Memorial Hospital, Thai Red Cross Society, Chulalongkorn University, Bangkok 10330, Thailand Department of Biochemistry, Osteoarthritis and Musculoskeleton Research Unit, Faculty of Medicine, King Chulalongkorn Memorial Hospital, Thai Red Cross Society, Chulalongkorn University, Bangkok 10330, Thailand Department of Orthopaedics, Vinai Parkpian Orthopaedic Research Center, Faculty of Medicine, King Chulalongkorn Memorial Hospital, Thai Red Cross Society, Chulalongkorn University, Bangkok 10330, Thailand Department of Orthopaedics, Vinai Parkpian Orthopaedic Research Center, Faculty of Medicine, King Chulalongkorn Memorial Hospital, Thai Red Cross Society, Chulalongkorn University, Bangkok 10330, Thailand Institute of Integrative Biology, University of Liverpool, Liverpool L69 7ZB, UK Institute of Integrative Biology, University of Liverpool, Liverpool L69 7ZB, UK Osteoarthritis (OA) is a degenerative articular disorder manifested by cartilage destruction, subchondral sclerosis, osteophytes, and synovitis, resulting in chronic joint pain and physical disability in the elderly. The purpose of this study was to investigate mitochondrial DNA copy number (mtDNA CN ) and inflammatory cytokines in primary knee OA patients and healthy volunteers. A total of 204 knee OA patients and 169 age-matched healthy volunteers were recruited. Their relative blood leukocyte mtDNA CN was assessed by quantitative real-time polymerase chain reaction (qRT-PCR), and ten inflammatory cytokines in their plasma were detected by multiplex immunoassay. Blood leukocyte mtDNA CN in the OA group was significantly lower than that in the control group. Leukocyte mtDNA CN in the control group was negatively correlated with their age ( r =−0.380, P < 0.0001), whereas mtDNA CN in the OA group was positively correlated with their age ( r =0.198, P < 0.001). Plasma interleukin-4 (IL-4) and IL-6 were significantly higher in the knee OA group than in the control group. The plasma IL-6 level was positively correlated with blood leukocyte mtDNA CN in the OA group ( r =0.547, P =0.0014). IL-5 showed as a major factor (coefficient 0.69) in the second dimension of principle components analysis (PCA)-transformed data and was significantly higher in the OA group ( P < 0.001) as well as negatively correlated with mtDNA CN ( r =−0.577, P < 0.001). These findings suggest that elevation of plasma IL-4 and IL-6 and a relative reduction in mtDNA CN might be effective biomarkers for knee OA. IL-5 is a plausible factor responsible for decreasing blood leukocyte mtDNA CN in knee OA patients. 目的 2 本实验研究全血白细胞线粒体DNA复制数量(mtDNA CN )和血浆炎性细胞因子在膝骨关节炎患者中的变化和其相关性. 创新点 2 探讨了老年(50~80岁)膝骨关节炎患者白细胞mtDNA CN 和血浆炎性细胞因子水平及二者的关系. 方法 2 分别收集膝骨关节炎组和对照组血液样本并对膝关节评分(Kellgren-Lawren grading).使用实时定量聚合酶链反应(qRT-PCR)检测相对mtDNA CN ;使用多重免疫分析(multiplex immunoassay)测定血浆中10种炎性细胞因子水平;应用线性相关、Logistic回归和主成分分析(PCA)揭示骨关节炎白细胞mtDNA CN 和血浆炎性细胞因子的相关性. 结论 2 血浆中白介素4(IL-4)、IL-6和全血白细胞mtDNA CN 可能是膝骨关节炎有效的生物标志物;IL-5则对mtDNA CN 减少具有潜在的影响. Inflammatory cytokine Blood leukocyte Knee Mitochondrial DNA copy number Osteoarthritis 炎性细胞因子 血液白细胞 膝关节 线粒体DNA复制数量 骨关节炎 Introduction 1 Osteoarthritis (OA) is a typical degenerative articular disorder resulting in chronic joint pain and physical disability in the elderly. It has been characterized by fibrillation, sclerosis, osteophyte formation, and progressive destruction of the articular cartilage (Manoy et al., 2018 2018 ). Multiple risk factors have been identified in the various stages of OA development such as gender, obesity, joint injury, senescence, and genetic factors. However, the etiology and pathogenesis of OA remain poorly understood. A number of biochemical and genetic factors have been documented as playing critical roles in the progression of OA. Mitochondria produce energy by synthesizing adenosine triphosphate (ATP) to drive normal cellular physiological functions. Mitochondrial DNA (mtDNA) does not contain introns or histones, and the oxidative damage level may be slightly lower in mtDNA than in nuclear DNA (Lim et al., 2005 2005 ). The fundamental mechanisms underlying mitochondrial dysfunction in OA involve increased chondrocyte apoptosis, decreased chondrocyte biosynthesis, cytokine-induced chondrocyte inflammation, and cartilage matrix calcification. Mitochondrial dysfunction in OA chondrocytes could stem from somatic mutations in the mtDNA or from the direct effects of proinflammatory cytokines and reactive oxygen species (ROS) (Blanco et al., 2011 2011 ). The mtDNA copy number (mtDNA CN ) decreases with age in adults over fifty, and thus mtDNA biogenesis is important to maintain an appropriate mtDNA CN content to slow down cellular aging (Dechsupa et al., 2017 2017 ). Recent studies have demonstrated that mtDNA CN was associated with osteoarthritis and might play a contributory role in the pathological process of OA (Fang et al., 2014 2014 ; Zhan and Honsawek, 2019 2019 ). A few inflammatory cytokines or chemokines are highly expressed in the circulation system of the elderly and maintain a low-grade systemic inflammatory condition (Hsu et al., 2009 2009 ; Schaap et al., 2009 2009 ; López-Otín et al., 2013 2013 ). Mitochondria and mtDNA content can influence low-grade systemic inflammation by various mechanisms. Systemic inflammation can be upgraded through weakening of the mitochondrial biogenesis of macrophages (Chawla et al., 2011 2011 ). Recently, leukocyte mtDNA CN was found to be negatively correlated with high-sensitivity C-reactive protein (hs-CRP), white blood cell count, and interleukin-6 (IL-6), and associated with oxidative stress in the elderly (Tanpaisankit et al., 2017 2017 ; Wu et al., 2017 2017 ). Inflammatory cytokines with high expression not only affect systemic and local conditions of OA, but also are crucial in synthesizing cartilage and regulating the extracellular matrix (Kapoor et al., 2011 2011 ; Mabey et al., 2016 2016 ). Both mtDNA content and knee OA are related to low-grade inflammatory reactions and the aging progress, and therefore it is necessary to investigate mtDNA CN and its relationships with inflammatory cytokines. The purpose of this study was to evaluate blood leukocyte mtDNA CN and inflammatory cytokines in patients with knee OA compared to those in a control group, and to determine the relationships between mtDNA CN and inflammatory cytokines in knee OA patients. Materials and methods 1 Study population 2 The study was approved by the Institutional Review Board (IRB number 565/59) on Human Research of the Faculty of Medicine, Chulalongkorn University, Thailand, and was conducted in compliance with the guidelines of the Declaration of Helsinki. All subjects gave written informed consent prior to their participation in this study. A total of 204 patients with unilateral primary knee OA (age range 50–80 years) were recruited in the OA group. OA patients were identified by diagnostic criteria of the American College of Rheumatology. Patients who had other chronic inflammatory diseases, immunological abnormalities, prior knee trauma, or knee surgery were excluded from the study. A total of 169 healthy volunteers (age range 50–80 years) with normal knee radiographs, without symptoms, signs, or a previous history of OA participated as the control group. The knee radiographs of controls and OA subjects were categorized using the Kellgren-Lawrence (KL) classification system (Kellgren and Lawrence, 1957 1957 ). The healthy volunteers had a KL grade of 0. Every patient included in the OA group had a KL grade equal to or greater than 2: 50 patients were Grade 2, 64 patients Grade 3, and 90 patients Grade 4. All patients were hospitalized at least one day before a knee arthroscopy or total knee replacement operation. The researchers were blinded as to which group each participant had been assigned. Serial numbers were randomly selected for labeling the biological samples from participants. Sample preparation 2 Whole blood samples collected into sodium heparin-coated tubes (Greiner Bio-one, Chonburi, Thailand) were centrifuged at 4000 g for 10 min to obtain blood leukocytes. Genomic DNA was then extracted from the leukocytes using an Illustra Blood Genomic Prep Mini Spin kit (GE Healthcare, Buckinghamshire, UK). The quality and concentration of the extracted DNA were identified using a NanoDrop 1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). The genomic DNA was aliquoted and stored at −80 °C until measurement. Among all participants, plasma samples of 12 healthy volunteers and 31 knee OA subjects were available for multiplex immunoassay. All plasma specimens were stored at −80 °C until further analysis. Measurement of blood leukocyte mtDNA CN 2 The relative mtDNA CN was determined using quantitative real-time polymerase chain reaction (qRT-PCR) as described previously (Xing et al., 2008 2008 ). DNA samples were amplified in 10-μL reactions using a Step One Plus Real Time PCR system (Applied Biosystems, Foster City, CA, USA). The primer sequences for the mitochondrial nicotinamide adenine dinucleotide (NADH) dehydrogenase 1 ( ND1 ) gene were: ND1 F, 5'-CCCTAAAACCCGCCACAT CT-3' and ND1 R, 5'-GAGCGATGGTGAGAGCTA AGGT-3'. The primer sequences for the nuclear human β-haemoglobin ( HGB ) gene were: HGB F, 5'-G TGCACCTGACTCCTGAGGAGA-3' and HGB R, 5'-CCTTGATACCAACCTGCCCAG-3'. Both reactions contained 5 μL qPCR Green Master Mixes (2×) (Biotech Rabbit, Germany), 2 μL DNA template (1.56 ng/mL), and 0.2 μL forward and reverse primers (10 μmol/L). The thermal cycling profile for both the ND1 and HGB genes started with 95 °C incubation for 30 s for 1 cycle, followed by 35 cycles of 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 50 s. All amplification specificity was regulated by melting curve analysis. Threshold cycle ( C T ) values were used to calculate the qRT-PCR results. An unchanged DNA sample was used as reference which was placed in the same well position of the tray. Each sample mtDNA CN was estimated by the comparative method using the fold induction 2 −ΔΔ C T equation (Livak and Schmittgen, 2001 2001 ). The Δ C T was determined by calculating the difference between the average C T value of the ND1 gene and that of the HGB gene. The difference between the sample Δ C T and reference Δ C T gave the ΔΔ C T value. Measurement of plasma cytokines 2 The levels of ten commonly tested cytokines in plasma were examined by magnetic bead-based multiplex measurement on 96-well plates (Bio-Plex Precision Pro, Bio-Rad, Hercules, USA). These 10-type beads were coupled by ten cytokine antibodies: IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α. Briefly, 50 μL of the coupled beads were added to the experimental wells. After washing, 50 μL of standards and plasma of OA patients or healthy volunteers were transferred into each well with beads and incubated for 1 h. Next, the detecting antibodies were added and incubated for 30 min. Streptavidin-phycoerythrin (streptavidin-PE) of appropriate concentration was added to each well and incubated for 10 min. Finally, assay buffer was added to resuspend the beads. A Bio-Plex 200 array reader (Bio-Plex 200 Multiplex System, Bio-Rad, Hercules, USA) was used to measure the levels of cytokines in suspension. Statistical analysis 2 All qualitative and quantitative variables were analyzed with software R Version 4.3.2 and GraphPad Prism (GraphPad Software Inc., La Jolla, CA, USA). Quantitative data are presented as mean±standard error of mean (SEM). Qualitative data are presented as frequencies. The normality of the quantitative data was identified by a Shapiro-Wilk test, and an F- test was used to check for homogeneity of the variance. Student’s t -test and Welch’s test were used for unpaired normal data based on the variance equality. Variables not normally distributed were analyzed by the Wilcoxon test. Qualitative data were analyzed by a Pearson χ 2 test to examine the frequency. Linear correlations of two variables were analyzed by Pearson’s correlation for normal data and Spearman’s correlation for non-normal data. Logistic regression analysis models were performed to determine the association between mtDNA CN and cytokines by taking age, gender, and body mass index (BMI) as the covariates. To investigate the interactions of multiple cytokines with mtDNA CN and disease, all detectable cytokines were integrated into different dimensions/components by applying principle components analysis (PCA) to reveal the potential variables. The number of dimensions was collected from evidence of scree plots and the Kaiser-Harris criterion for eigenvalues greater than 1. A non-rotation method was used in PCA to display data features. Interpretation of each dimension was supported by a high absolute value of eigenvalue loading coefficients. The calculation of individual dimension scores for each participant was based on detectable inflammatory cytokines. Differences between the control and OA groups in each dimension were analyzed with these scores. Subsequently, the correlations between standardized mtDNA CN (mean=0, standard deviation (SD)=1) and individual scores of each dimension in different groups were analyzed using a simple linear model. P < 0.05 was considered to indicate a significant difference. Results 1 Clinical characteristics of the participants 2 Demographic data of the patients included in this study are displayed in Table 1 1 . There were no significant age differences between groups ( P =0.132). The number of females was significantly higher in the OA group than in the control group ( P < 0.001). The mean BMI was significantly lower in the control group than in the OA group ( P < 0.001). OA: osteoarthritis; BMI: body mass index; KL: Kellgren-Lawrence. Data are expressed as mean±standard error of mean (SEM), number (percentage of female), or number 2 Group 2 Age (year) 2 Female/male 2 BMI (kg/m 2 ) 3 KL grade 2 3 4 Control ( n =169) 62.69±0.42 111/58(65.68%) 25.12±0.34 OA ( n =204) 63.70±0.52 168/36(82.35%) 27.09±0.26 50 64 90 7 P -value 0.132 <0.001 <0.001 Correlation of mtDNA CN and age in patients with knee OA 2 The mean blood leukocyte mtDNA CN of the OA group was significantly lower than that of the control group (0.50±0.03 vs. 1.07±0.04, P < 0.0001; Fig. 1 1 ). There were no significant differences in mtDNA CN among various KL grades of the OA group ( P =0.3277). However, the blood leukocyte mtDNA CN of OA patients with KL Grade 2, 3, or 4 was significantly lower than that of controls ( P < 0.0001; Fig. 1 1 ). In the control group, the mtDNA CN was negatively correlated with the age of healthy participants ( r = −0.380; P < 0.0001). However, the mtDNA CN increased with age in the OA group ( r =0.198, P =0.0025). Thus, the relationships between age and mtDNA CN showed opposing linear correlations in the control and OA groups. The relative mtDNA CN of blood leukocytes of the OA group was lower than that of the control group ( P < 0.0001). The blood leukocyte mtDNA CN of OA patients with KL Grade 2, 3, or 4 was significantly lower than that of controls ( P < 0.0001). The relative mtDNA CN of blood leukocytes did not differ among KL Grade 2, 3, and 4 patients in the OA group ( P > 0.05). *** P < 0.0001 vs. control Inflammatory cytokine levels in plasma 2 Plasma samples from 12 healthy volunteers and 31 OA patients were used to assess the levels of inflammatory cytokines. The levels of two cytokines, IL-4 and IL-6, were significantly greater in the OA patients than in the controls ( P =0.003 and P < 0.001, respectively; Table 2 2 ). The levels of IL-1β, IL-2, IL-5, IL-10, IL-12p70, IL-13, and TNF-α were higher in the OA group, but the differences were not significant. Plasma IFN-γ was undetectable in the control group, but was detected in four participants in the OA group. CI: confidence interval; ND: not detectable; NA: not available. Data are expressed as mean±standard error of mean (SEM). IFN-γ was detectable in only four knee osteoarthritis (OA) patients 2 Cytokine 2 Concentration (pg/mL) * * 2 95% CI 2 P -value Control group ( n =12) OA group ( n =31)  IL-1β 20.550±1.666 23.354±1.724 −8.802, 3.195 0.351  IL-2 115.492±9.592 138.937±9.358 −56.257, 9.367 0.157  IL-4 2.087±0.408 4.072±0.378 −2.840, −0.530 0.003  IL-5 117.155±8.996 148.521±15.710 −49.800, 4.820 0.088  IL-6 156.740±12.953 437.826±77.224 −328.160, −73.660 <0.001  IL-10 131.324±10.341 157.557±12.856 −59.633, 7.167 0.120  IL-12p70 2.817±0.501 6.173±1.249 −4.870, 0.240 0.155  IL-13 24.658±2.025 27.539±2.032 −9.981, 4.217 0.417  IFN-γ ND 5.385±1.061 a a NA NA  TNF-α 8.578±0.788 9.576±0.759 −9.981, 4.217 0.454 Correlation of cytokines and mtDNA CN 2 Since ten paired inflammatory cytokine levels and mtDNA CN were acquired from 43 participants, a matrix correlation analysis was applied to interpret their mutual relationship. The correlogram of the control group is shown in Fig. 2a 2a . Blue circles demonstrate that most inflammatory cytokines in the control group were positively correlated with other cytokines ( P < 0.05). In the OA group, only IL-6 was positively correlated with mtDNA CN ( r =0.547, P =0.0014; Fig. 2b 2b ). Blank squares represent undiscovered relationships between each cytokine and mtDNA CN . In the OA group, there were no associations between IL-6 and other inflammatory cytokines, except for IL-1β ( r =0.361, P =0.0458). The other cytokines showed positive associations with each other. As age, gender, and BMI can influence the findings, we performed logistic regression analyses to determine the correlation between plasma cytokines and blood leukocyte mtDNA CN of OA patients by taking age, gender, and BMI as the covariates. We observed no significant correlation between blood leukocyte mtDNA CN and plasma cytokines in the knee OA patients. (a) Correlogram of the control group; (b) Correlogram of the osteoarthritis (OA) group. The colored gradient legends represent coefficients of correlation r values from +1.0 (dark blue) to −1.0 (dark red). The presence of a circle indicates a significant correlation ( P < 0.05); conversely, blank squares indicate no significance ( P > 0.05). All coefficients were computed by Spearman’s rho rank correlation for possible pairs of variables in the matrix Principle components analysis 2 Eigenvalues of the three dimensions were greater than 1; the first dimension had an eigenvalue of 5.47, the second 1.11, and the third 1.06 (Fig. 3 3 ). These three dimensions accounted for 60.8%, 12.3%, and 11.7% of the variance for the nine cytokine variables, respectively, and all three cumulative dimensions accounted for 84.5% of the variance in the height variables (Table 3 3 ). The eigenvalues of dimensions 1, 2, and 3 were more than 1 IL: interleukin; TNF: tumor necrosis factor; OA: osteoarthritis. Data are expressed as mean±standard error of mean (SEM) 2 Dimension 2 2 Eigenvalue 2 2 Explained proportion 13 Scoring coefficient IL-1β 2 IL-2 2 IL-4 2 IL-5 2 IL-6 2 IL-10 2 IL-12p70 1 2 5.47 2 60.8% 0.93 2 0.94 2 0.69 2 0.40 2 0.06 2 0.94 2 0.62 2 2 1.11 2 12.3% −0.23 2 −0.23 2 0.46 2 0.69 2 0.24 2 −0.04 2 0.42 3 2 1.06 2 11.7% 0.01 2 0.02 2 0.11 2 −0.40 2 0.94 2 0.04 2 0.00 18 18 3 Dimension 3 Scoring coefficient 7 Comparison in 3 major dimensions 7 Correlation with standardized mtDNA CN 2 IL-13 2 2 TNF-α 3 2 Control group * * 2 2 OA group * * 2 2 P -value 4 Control group 3 OA group 2 r 2 P -value 2 r P -value 18 1 0.95 2 0.95 3 0.876±0.437 2 −0.339±0.454 2 0.1280 2 0.004 2 0.9893 2 −0.0860 0.6439 2 −0.23 2 −0.19 3 −0.545±0.061 2 0.283±0.209 2 0.0007 2 0.008 2 0.9936 2 −0.5767 0.0007 3 −0.03 2 −0.02 3 −0.453±0.029 2 0.175±0.210 2 <0.0001 2 −0.280 2 0.5272 2 0.1657 0.3730 All nine detectable cytokines were positively correlated with the first dimension, which suggested that the nine variables worked together to produce the effects seen in OA. The first dimension was highly correlated with IL-13, TNF-α, IL-2, IL-10, and IL-1β whose correlation coefficients were more than 0.90. Therefore, this dimension was a predominant measurement for these five cytokines. Although correlation coefficients for IL-4 and IL-12p70 were more than 0.5, they were not considered to be the main variables in the first dimension. As the coefficient value was just 0.06 for IL-6, this dimension could not be regarded as a measure of IL-6. There were no statistical differences between the OA and the control groups in the first dimension. Also, there were no significant correlations with standardized mtDNA CN in either group (Table 3 3 ). In the second dimension, the coefficient for IL-5 increased to 0.69, and four cytokines (IL-5, IL-4, IL-12p70, and IL-6) were then positively correlated with this dimension (Table 3 3 , Fig. 4a 4a ). The mean individual score for the OA group was higher than that of the control group in the second dimension. The standardized mtDNA CN was negatively correlated with the individual scores in the second dimension for the OA group ( r =−0.577, P < 0.001), but there was no significant correlation for the control group. Participants’ component scores were plotted against each other and in group-dependent clusters: the principle components analysis (PCA) biplot for dimensions 1 versus 2 (a) and dimensions 1 versus 3 (b). Dim: dimension; Ctrl: control; OA: osteoarthritis In the third dimension, IL-6 became the only predominant factor, with a coefficient value of 0.94. In contrast, IL-5 was negatively correlated to this dimension, with a coefficient value of 0.40 (Table 3 3 , Fig. 4b 4b ). Other values were less than or equal to 0.11. The mean score of the control group was significantly different to that of the OA group ( P < 0.0001). There was no significant correlation between the cytokines studied and the standardized mtDNA CN for each group (Table 3 3 ). Discussion 1 Few studies have described the mtDNA content from peripheral blood leukocytes of patients with primary knee OA. Our study demonstrated that blood leukocyte mtDNA CN was lower in OA patients than in healthy participants. We found that IL-4 and IL-6 concentrations were significantly higher in OA patients. Moreover, IL-6 was significantly and positively correlated with mtDNA CN in OA patients. Results from the PCA indicated that underlying dimensions of multiple cytokines and mtDNA CN affected knee OA. Blood leukocyte mtDNA CN was higher in knee OA patients and inversely correlated to dimension 2 in which IL-5 was the major factor. IL-5 was higher in patients with knee OA. In dimension 3, IL-6 was the major factor and IL-6 levels were higher in the OA group. These results suggest that these cytokines may contribute to the biological characteristics of primary OA and may cause chronic low-grade inflammation. Abnormal mtDNA CN can disrupt the gene expression, differentiation, and migration of normal cells (Liu et al., 2015 2015 ). Mitochondria dysfunction of peripheral leucocytes may be associated with OA development by increasing ROS and apoptosis (Meyer et al., 2013 2013 ; Yang et al., 2013 2013 ). Previous studies found that leukocyte mtDNA was lower in patients with various diseases including metabolic syndrome, cancer, and neurodegenerative diseases (Chomyn and Attardi, 2003 2003 ). Fang et al. ( 2014 2014 ) reported that mtDNA CN was higher in knee OA subjects compared to controls, but the mtDNA CN did not differ among various subgroups of OA subjects. The explanations for these conflicting results remain unclear, but might be due to differences in clinical settings, disease advancement, populations, ethnic groups, or the assays applied. Age is an influential factor for mtDNA CN . In the general population, mtDNA content and age show a non-linear relationship; the mtDNA content of people over 50 years old has been shown to decrease with their age (Dechsupa et al., 2017 2017 ). Our findings from the healthy volunteers also showed that age was negatively correlated with relative mtDNA CN . Inflammation has been recognized as a contributor to the symptoms and progression of OA, especially in cartilage and the synovium (Smith et al., 1997 1997 ; Blanco et al., 2011 2011 ). Cytokines and mediators of local inflammatory events involve circulating blood. Numerous studies reported that several inflammation-related cytokines were higher in OA blood (Attur et al., 2012 2012 ; Berenbaum, 2013 2013 ; Mabey et al., 2016 2016 ). Low-grade, systemic, and chronic inflammation might be a pivotal sign or factor for initiating or developing the disorder (Blanco et al., 2018 2018 ). IL-6 is a pro-inflammatory cytokine which decreases type II collagen and increases matrix metalloproteinases (Porée et al., 2008 2008 ; Kapoor et al., 2011 2011 ). Higher concentrations of IL-6 have been found in plasma and sera of OA patients (Kaneko et al., 2000 2000 ; Mabey et al., 2016 2016 ). In a 15-year follow-up study, researchers discovered that IL-6 was consistently upregulated in patients with radiographic knee OA (Livshits et al., 2009 2009 ). In contrast, lower IL-6 levels have been found to decrease the probability of development into OA by 87.01% (Goekoop et al., 2010 2010 ). IL-6 is also recognized as a myokine, which increases by up to 100-fold compared to the baseline level when skeletal muscle contracts during aerobic exercise. IL-6 may be acutely secreted and have an anti-inflammatory effect (Pedersen and Febbraio, 2008 2008 ). Prolonged higher levels of IL-6 in the blood can maintain low-grade, chronic, and systemic inflammation in OA patients instead of the benefits of acute IL-6 production by myocytes. As an anti-inflammatory cytokine, IL-4 regulates macrophage action to decrease inflammation, regulate lipid accretion, inhibit the expression of a few pro-inflammatory cytokines, and increase glucose tolerance (Chang et al., 2012 2012 ). IL-4 levels were higher in plasma of the OA group in this and our previous studies (Mabey et al., 2016 2016 ). However, the serum IL-4 concentration was lower in the OA model of a rat study (Guo et al., 2015 2015 ). These contrasting results were found in real cases of knee OA and in an animal model of OA using an identical IL-4 epitope recognized by serum IL-4 receptor and antibodies of the immunoassay. More importantly, blood IL-4 levels vary with different stages of knee OA: the IL-4 level of early knee OA (EOA) is higher than that of advanced knee OA (AOA) (Barker et al., 2014 2014 ). We speculate that low-grade systemic inflammation of OA induces IL-4 release into circulating blood in EOA. However, increased IL-4 promotes an increase in soluble IL-4 receptor (IL-4R) to maintain a balanced IL-4/IL-4R system (Silvestri et al., 2006 2006 ). The advantage of IL-4 effects can be blocked by increasing the levels of soluble IL-4 receptor which down-regulates IL-4 activity and quantity in AOA. Type 2 helper T cells and mast cells express IL-5 to induce eosinophil activation, adhesion, chemotaxis, and the release of other inflammatory cytokines and chemokines (Ashraf et al., 2015 2015 ). Vangsness et al. ( 2011 2011 ) reported that the amount of IL-5 in synovial fluid (SF) in the knee was higher in AOA patients, according to the International Cartilage Repair Society (ICRS) classification. Six cytokine genes, IL-4 , IL-13 , IL-5 , IL-9 , colony stimulating factor 2 ( CSF2 ), and IL-3 , are located close together in the 5q31–33 region of the human genome, forming a cytokine cluster (Thomas et al., 1997 1997 ). Thus, variation in IL-5 could be influenced by IL-4 gene expression. This was confirmed by a report that IL-5 production could be down-regulated in CD4 + T cells of IL-4 gene knock-out mice (Kopf et al., 1993 1993 ). Our study applied PCA to identify patterns within complicated biological data. The mtDNA CN has been negatively correlated with CRP-related factors such as IL-6, fibrinogen, leukocyte count, and hs-CRP in the elderly (Wu et al., 2017 2017 ). Although our research did not find any correlation between leukocyte mtDNA CN and plasma IL-6 level in healthy volunteers, IL-6 was positively correlated with mtDNA CN in elderly patients with knee OA. The impact of IL-6 on mtDNA CN probably was influenced by IL-5. According to dimension 3 of the PCA, the scoring coefficient of IL-5 was −0.40, whereas that of IL-6 was 0.94. When IL-5 became the largest positive coefficient (0.69) in dimension 2, it was negatively correlated with blood leukocyte mtDNA CN in the OA group. Yousefi et al. ( 2008 2008 ) revealed that, after stimulation of eotaxin, lipopolysaccharide (LPS), or complement factor 5a (C5a), IL-5-pre-treated eosinophils were capable of releasing mtDNA into extracellular areas resulting in a decline in intracellular mtDNA. Moreover, this process was dependent on ROS. The current study had several limitations that should be noted. First, the sampling of a relatively small number of subjects in one single site cannot represent the general population. Larger scale, multicenter studies should be conducted to verify our conclusions. Second, the mtDNA CN of all participants from the controls and OA participants has been investigated, but the limited availability of plasma inflammatory cytokine data from controls and knee OA patients posed significant challenges in the study. Another caveat is the lack of data regarding in vitro cell culture models of human chondrocytes. Further studies of mtDNA CN and cytokines in human primary chondrocytes obtained from healthy control and OA subjects will be useful for validating the findings of this study. Lastly, the cross-sectional design prevented determination of cause-and-effect relationships, and the potential for confounding variables needs to be taken into consideration. In conclusion, blood leukocyte mtDNA CN in the knee OA subjects was significantly lower than that in the controls. Plasma IL-4 and IL-6 were significantly greater in the knee OA cases than in the controls. The plasma IL-6 level was positively correlated with blood leukocyte mtDNA CN in knee OA. PCA showed that IL-5 was a major factor and was significantly higher in the OA patients and negatively correlated with blood leukocyte mtDNA CN . High levels of IL-4 and IL-6 are potential biomarkers for OA disease diagnosis and pathophysiology. IL-5 could be responsible for the decline in blood leukocyte mtDNA CN in primary knee OA. References MI Ashraf , M Shahzad , A Shabbir . Oxyresveratrol ameliorates allergic airway inflammation via attenuation of IL-4, IL-5, and IL-13 expression levels . Cytokine , 2015 . 76 ( 2 ): 375 - 381 . 26431781 DOI: 10.1016/j.cyto.2015.09.013 http://doi.org/10.1016/j.cyto.2015.09.013 . Oxyresveratrol ameliorates allergic airway inflammation via attenuation of IL-4, IL-5, and IL-13 expression levels M Attur , A Statnikov , CF Aliferis , 等 . Inflammatory genomic and plasma biomarkers predict progression of symptomatic knee OA (SKOA) . Osteoarthritis Cartilage , 2012 . 20 ( Suppl 1 ): S34 - S35 . DOI: 10.1016/j.joca.2012.02.562 http://doi.org/10.1016/j.joca.2012.02.562 . Inflammatory genomic and plasma biomarkers predict progression of symptomatic knee OA (SKOA) T Barker , VE Rogers , VT Henriksen , 等 . Serum cytokines are increased and circulating micronutrients are not altered in subjects with early compared to advanced knee osteoarthritis . Cytokine , 2014 . 68 ( 2 ): 133 - 136 . 24787465 DOI: 10.1016/j.cyto.2014.04.004 http://doi.org/10.1016/j.cyto.2014.04.004 . Serum cytokines are increased and circulating micronutrients are not altered in subjects with early compared to advanced knee osteoarthritis F Berenbaum . Osteoarthritis as an inflammatory disease (osteoarthritis is not osteoarthrosis!) . Osteoarthritis Cartilage , 2013 . 21 ( 1 ): 16 - 21 . 23194896 DOI: 10.1016/j.joca.2012.11.012 http://doi.org/10.1016/j.joca.2012.11.012 . Osteoarthritis as an inflammatory disease (osteoarthritis is not osteoarthrosis!) FJ Blanco , I Rego , C Ruiz-Romero . The role of mitochondria in osteoarthritis . Nat Rev Rheumatol , 2011 . 7 ( 3 ): 161 - 169 . 21200395 DOI: 10.1038/nrrheum.2010.213 http://doi.org/10.1038/nrrheum.2010.213 . The role of mitochondria in osteoarthritis FJ Blanco , AM Valdes , I Rego-Pérez . Mitochondrial DNA variation and the pathogenesis of osteoarthritis phenotypes . Nat Rev Rheumatol , 2018 . 14 ( 6 ): 327 - 340 . 29670212 DOI: 10.1038/s41584-018-0001-0 http://doi.org/10.1038/s41584-018-0001-0 . Mitochondrial DNA variation and the pathogenesis of osteoarthritis phenotypes YH Chang , KT Ho , SH Lu , 等 . Regulation of glucose/lipid metabolism and insulin sensitivity by interleukin-4 . Int J Obes (Lond) , 2012 . 36 ( 7 ): 993 - 998 . 21894160 DOI: 10.1038/ijo.2011.168 http://doi.org/10.1038/ijo.2011.168 . Regulation of glucose/lipid metabolism and insulin sensitivity by interleukin-4 A Chawla , KD Nguyen , YPS Goh . Macrophage-mediated inflammation in metabolic disease . Nat Rev Immunol , 2011 . 11 ( 11 ): 738 - 749 . 21984069 DOI: 10.1038/nri3071 http://doi.org/10.1038/nri3071 . Macrophage-mediated inflammation in metabolic disease A Chomyn , G Attardi . MtDNA mutations in aging and apoptosis . Biochem Biophys Res Commun , 2003 . 304 ( 3 ): 519 - 529 . 12729587 DOI: 10.1016/s0006-291x(03)00625-9 http://doi.org/10.1016/s0006-291x(03)00625-9 . MtDNA mutations in aging and apoptosis S Dechsupa , W Singhatanadgige , W Limthongkul , 等 . Alterations of relative telomere length and mitochondrial DNA copy number from ligamentum flavum-derived cells in lumbar spinal stenosis: pilot study . Chula Med J , 2017 . 61 ( 4 ): 497 - 509 . . Alterations of relative telomere length and mitochondrial DNA copy number from ligamentum flavum-derived cells in lumbar spinal stenosis: pilot study HZ Fang , XW Liu , LJ Shen , 等 . Role of mtDNA haplogroups in the prevalence of knee osteoarthritis in a southern Chinese population . Int J Mol Sci , 2014 . 15 ( 2 ): 2646 - 2659 . 24534808 DOI: 10.3390/ijms15022646 http://doi.org/10.3390/ijms15022646 . Role of mtDNA haplogroups in the prevalence of knee osteoarthritis in a southern Chinese population RJ Goekoop , M Kloppenburg , HM Kroon , 等 . Low innate production of interleukin-1β and interleukin-6 is associated with the absence of osteoarthritis in old age . Osteoarthritis Cartilage , 2010 . 18 ( 7 ): 942 - 947 . 20417290 DOI: 10.1016/j.joca.2010.03.016 http://doi.org/10.1016/j.joca.2010.03.016 . Low innate production of interleukin-1β and interleukin-6 is associated with the absence of osteoarthritis in old age SY Guo , YJ Ding , L Li , 等 . Correlation of CD 4 + CD 25 + Foxp 3 + Treg with the recovery of joint function after total knee replacement in rats with osteoarthritis . Genet Mol Res , 2015 . 14 ( 3 ): 7290 - 7296 . 26214407 DOI: 10.4238/2015.July.3.4 http://doi.org/10.4238/2015.July.3.4 . Correlation of CD4+ CD25+ Foxp3+ Treg with the recovery of joint function after total knee replacement in rats with osteoarthritis FC Hsu , SB Kritchevsky , YM Liu , 等 . Association between inflammatory components and physical function in the health, aging, and body composition study: a principal component analysis approach . J Gerontol A Biol Sci Med Sci , 2009 . 64A ( 5 ): 581 - 589 . DOI: 10.1093/gerona/glp005 http://doi.org/10.1093/gerona/glp005 . Association between inflammatory components and physical function in the health, aging, and body composition study: a principal component analysis approach S Kaneko , T Satoh , J Chiba , 等 . Interleukin-6 and interleukin-8 levels in serum and synovial fluid of patients with osteoarthritis . Cytokines Cell Mol Ther , 2000 . 6 ( 2 ): 71 - 79 . 11108572 DOI: 10.1080/13684730050515796 http://doi.org/10.1080/13684730050515796 . Interleukin-6 and interleukin-8 levels in serum and synovial fluid of patients with osteoarthritis M Kapoor , J Martel-Pelletier , D Lajeunesse , 等 . Role of proinflammatory cytokines in the pathophysiology of osteoarthritis . Nat Rev Rheumatol , 2011 . 7 ( 1 ): 33 - 42 . 21119608 DOI: 10.1038/nrrheum.2010.196 http://doi.org/10.1038/nrrheum.2010.196 . Role of proinflammatory cytokines in the pathophysiology of osteoarthritis JH Kellgren , JS Lawrence . Radiological assessment of osteo-arthrosis . Ann Rheum Dis , 1957 . 6 ( 4 ): 494 - 502 . DOI: 10.1136/ard.16.4.494 http://doi.org/10.1136/ard.16.4.494 . Radiological assessment of osteo-arthrosis M Kopf , G le Gros , M Bachmann , 等 . Disruption of the murine IL-4 gene blocks Th2 cytokine responses . Nature , 1993 . 362 ( 6417 ): 245 - 248 . 8384701 DOI: 10.1038/362245a0 http://doi.org/10.1038/362245a0 . Disruption of the murine IL-4 gene blocks Th2 cytokine responses KS Lim , K Jeyaseelan , M Whiteman , 等 . Oxidative damage in mitochondrial DNA is not extensive . Ann N Y Acad Sci , 2005 . 1042 ( 1 ): 210 - 220 . 15965065 DOI: 10.1196/annals.1338.023 http://doi.org/10.1196/annals.1338.023 . Oxidative damage in mitochondrial DNA is not extensive SF Liu , HC Kuo , CW Tseng , 等 . Leukocyte mitochondrial DNA copy number is associated with chronic obstructive pulmonary disease . PLoS ONE , 2015 . 10 ( 9 ): e0138716 26394041 DOI: 10.1371/journal.pone.0138716 http://doi.org/10.1371/journal.pone.0138716 . Leukocyte mitochondrial DNA copy number is associated with chronic obstructive pulmonary disease KJ Livak , TD Schmittgen . Analysis of relative gene expression data using real-time quantitative PCR and the 2 −ΔΔ C T method . Methods , 2001 . 25 ( 4 ): 402 - 408 . 11846609 DOI: 10.1006/meth.2001.1262 http://doi.org/10.1006/meth.2001.1262 . Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method G Livshits , GJ Zhai , DJ Hart , 等 . Interleukin-6 is a significant predictor of radiographic knee osteoarthritis: the Chingford study . Arthritis Rheum , 2009 . 60 ( 7 ): 2037 - 2045 . 19565477 DOI: 10.1002/art.24598 http://doi.org/10.1002/art.24598 . Interleukin-6 is a significant predictor of radiographic knee osteoarthritis: the Chingford study C López-Otín , MA Blasco , L Partridge , 等 . The hallmarks of aging . Cell , 2013 . 153 ( 6 ): 1194 - 1217 . 23746838 DOI: 10.1016/j.cell.2013.05.039 http://doi.org/10.1016/j.cell.2013.05.039 . The hallmarks of aging T Mabey , S Honsawek , A Tanavalee , 等 . Plasma and synovial fluid inflammatory cytokine profiles in primary knee osteoarthritis . Biomarkers , 2016 . 21 ( 7 ): 639 - 644 . 27122451 DOI: 10.3109/1354750X.2016.1171907 http://doi.org/10.3109/1354750X.2016.1171907 . Plasma and synovial fluid inflammatory cytokine profiles in primary knee osteoarthritis P Manoy , W Anomasiri , P Yuktanandana , 等 . Relationship of serum leptin and 25-hydroxyvitamin D in knee osteoarthritis patients . Chula Med J , 2018 . 62 ( 6 ): 1037 - 1047 . . Relationship of serum leptin and 25-hydroxyvitamin D in knee osteoarthritis patients A Meyer , J Zoll , AL Charles , 等 . Skeletal muscle mitochondrial dysfunction during chronic obstructive pulmonary disease: central actor and therapeutic target . Exp Physiol , 2013 . 98 ( 6 ): 1063 - 1078 . 23377494 DOI: 10.1113/expphysiol.2012.069468 http://doi.org/10.1113/expphysiol.2012.069468 . Skeletal muscle mitochondrial dysfunction during chronic obstructive pulmonary disease: central actor and therapeutic target BK Pedersen , MA Febbraio . Muscle as an endocrine organ: focus on muscle-derived interleukin-6 . Physiol Rev , 2008 . 88 ( 4 ): 1379 - 1406 . 18923185 DOI: 10.1152/physrev.90100.2007 http://doi.org/10.1152/physrev.90100.2007 . Muscle as an endocrine organ: focus on muscle-derived interleukin-6 B Porée , M Kypriotou , C Chadjichristos , 等 . Interleukin-6 (IL-6) and/or soluble IL-6 receptor down-regulation of human type II collagen gene expression in articular chondrocytes requires a decrease of Sp1·Sp3 ratio and of the binding activity of both factors to the COL2A1 promoter . J Biol Chem , 2008 . 283 ( 8 ): 4850 - 4865 . 18065760 DOI: 10.1074/jbc.M706387200 http://doi.org/10.1074/jbc.M706387200 . Interleukin-6 (IL-6) and/or soluble IL-6 receptor down-regulation of human type II collagen gene expression in articular chondrocytes requires a decrease of Sp1·Sp3 ratio and of the binding activity of both factors to the COL2A1 promoter LA Schaap , SMF Pluijm , DJH Deeg , 等 . Higher inflammatory marker levels in older persons: associations with 5-year change in muscle mass and muscle strength . J Gerontol A Biol Sci Med Sci , 2009 . 64A ( 11 ): 1183 - 1189 . DOI: 10.1093/gerona/glp097 http://doi.org/10.1093/gerona/glp097 . Higher inflammatory marker levels in older persons: associations with 5-year change in muscle mass and muscle strength T Silvestri , L Pulsatelli , P Dolzani , 等 . Elevated serum levels of soluble interleukin-4 receptor in osteoarthritis . Osteoarthritis Cartilage , 2006 . 14 ( 7 ): 717 - 719 . 16647277 DOI: 10.1016/j.joca.2006.02.015 http://doi.org/10.1016/j.joca.2006.02.015 . Elevated serum levels of soluble interleukin-4 receptor in osteoarthritis MD Smith , S Triantafillou , A Parker , 等 . Synovial membrane inflammation and cytokine production in patients with early osteoarthritis . J Rheumatol , 1997 . 24 ( 2 ): 365 - 371 . 9034998 . Synovial membrane inflammation and cytokine production in patients with early osteoarthritis M Tanpaisankit , C Hongsaprabhas , C Charoenlap , 等 . High oxidative stress and decrease of mitochondrial DNA copies in musculoskeletal tumors . Chula Med J , 2017 . 61 ( 6 ): 771 - 782 . . High oxidative stress and decrease of mitochondrial DNA copies in musculoskeletal tumors NS Thomas , J Wilkinson , ST Holgate . The candidate region approach to the genetics of asthma and allergy . Am J Respir Crit Care Med , 1997 . 156 ( 4 ): S144 - S151 . 9351596 DOI: 10.1164/ajrccm.156.4.12-tac-13 http://doi.org/10.1164/ajrccm.156.4.12-tac-13 . The candidate region approach to the genetics of asthma and allergy Jr CT Vangsness , WS Burke , SJ Narvy , 等 . Human knee synovial fluid cytokines correlated with grade of knee osteoarthritis–a pilot study . Bull NYU Hosp Jt Dis , 2011 . 69 ( 2 ): 122 - 127 . 22035391 . Human knee synovial fluid cytokines correlated with grade of knee osteoarthritis–a pilot study IC Wu , CC Lin , CS Liu , 等 . Interrelations between mitochondrial DNA copy number and inflammation in older adults . J Gerontol A Biol Sci Med Sci , 2017 . 72 ( 7 ): 937 - 944 . 28329339 DOI: 10.1093/gerona/glx033 http://doi.org/10.1093/gerona/glx033 . Interrelations between mitochondrial DNA copy number and inflammation in older adults JL Xing , M Chen , CG Wood , 等 . Mitochondrial DNA content: its genetic heritability and association with renal cell carcinoma . J Natl Cancer Inst , 2008 . 100 ( 15 ): 1104 - 1112 . 18664653 DOI: 10.1093/jnci/djn213 http://doi.org/10.1093/jnci/djn213 . Mitochondrial DNA content: its genetic heritability and association with renal cell carcinoma YH Yang , AV Bazhin , J Werner , 等 . Reactive oxygen species in the immune system . Int Rev Immunol , 2013 . 32 ( 3 ): 249 - 270 . 23617726 DOI: 10.3109/08830185.2012.755176 http://doi.org/10.3109/08830185.2012.755176 . Reactive oxygen species in the immune system S Yousefi , JA Gold , N Andina , 等 . Catapult-like release of mitochondrial DNA by eosinophils contributes to antibacterial defense . Nat Med , 2008 . 14 ( 9 ): 949 - 953 . 18690244 DOI: 10.1038/nm.1855 http://doi.org/10.1038/nm.1855 . Catapult-like release of mitochondrial DNA by eosinophils contributes to antibacterial defense D Zhan , S Honsawek . Reduction of leukocyte mitochondrial DNA copy number in knee osteoarthritis . Chula Med J , 2019 . 63 ( 3 ): 207 - 209 . . Reduction of leukocyte mitochondrial DNA copy number in knee osteoarthritis Compliance with ethics guidelines Dong ZHAN, Aree TANAVALEE, Saran TANTAVISUT, Srihatach NGARMUKOS, Steven W. EDWARDS, and Sittisak HONSAWEK declare that they have no conflict of interest. The study protocol conformed to the ethical standards outlined in the Declaration of Helsinki of 1975, as revised in 2008 (5) and was approved by the Ethical Committee on Human Research of the Faculty of Medicine, Chulalongkorn University, Thailand. All study subjects were fully informed of the study protocol and procedures prior to participating in the study. Written informed consent was obtained from all participants before any procedures were performed. 10.1631/jzus.B1900352 Project supported by the 90th Anniversary of Chulalongkorn University (No. RA61/104), the Research Chair Grant from the National Science and Technology Development Agency, the 100th Anniversary of Chulalongkorn University Fund for Doctoral Scholarship, and the Overseas Research Experience Scholarship for Graduate Student, Thailand and China Scholarship Council, China 2019-06-27 2019-09-08 2022-01-05 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2020 2020 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 01 21