Diagnostic Value of Interleukin-34 as a Novel Biomarker for Severity of Knee Osteoarthritis

Abstract

Objectives

This study aimed to determine whether plasma and synovial fluid interleukin-34 (IL-34), an inflammatory cytokine reportedly implicated in synovial inflammation-induced joint degeneration, were associated with radiographic severity of knee osteoarthritis (OA) patients and could emerge as knee OA biomarkers.

Design

Ninety-six knee OA patients and 72 healthy controls were recruited. Plasma and synovial fluid IL-34 levels were quantified using ELISA. IL-34 mRNA and protein expressions in inflamed (n = 15) and noninflamed synovial tissues (n = 15) of knee OA patients were determined using real-time polymerase chain reaction and immunohistochemistry, respectively.

Results

Significant increases in plasma and synovial fluid IL-34 levels were found in knee OA patients—especially those with advanced stage (P < 0.001, P < 0.001, respectively). Both plasma and synovial fluid IL-34 levels were positively associated with radiographic severity (r = 0.64, P < 0.001; r = 0.50, P < 0.001, respectively). There was a direct link between plasma and synovial fluid IL-34 (r = 0.64, P < 0.001). Receiver operating characteristic curve analysis uncovered that the optimal cutoff value of plasma IL-34 as a novel biomarker reflecting knee OA severity was defined at 3750.0 pg/mL (AUC = 0.85), with a sensitivity of 83.1% and a specificity of 74.2%. Further analysis revealed that IL-34 mRNA expression was significantly upregulated in inflamed synovium compared with noninflamed synovium obtained from knee OA patients (P < 0.001), consistent with protein expression analysis demonstrating IL-34 overexpression localized in the lining and sublining layers of inflamed synovium.

Conclusions

All findings suggest that elevated plasma and synovial fluid IL-34 would reflect knee OA severity and might have potential utility as biomarkers for the disease progression.

Introduction

Knee osteoarthritis (OA) is a chronic degenerative joint disease and the most common cause of disability in the elderly, which is becoming a major public health problem. ¹ Although the specific order of pathologic alterations occurring in knee OA is far less clear, synovial inflammation has been recognized as the cornerstone event of knee OA. ² This pathologic feature leads to debilitating and irreversible joint destruction in the patients, through which a number of pro-inflammatory cytokines and mediators secreted by resident joint and infiltrating immune cells play an important role in cartilage degradation and excessive bone remodeling.^3,4 Along with the synovium, the infrapatellar fat pad (IFP), an adipose tissue near synovium, reportedly secreted various cytokines and adipokines, thus playing an critical role in an inflammatory response contributing to the developmental knee OA.^5,6 Despite extensive research efforts to identify and develop disease markers and effective treatments, currently available approaches could not stop the progression of synovitis-induced joint degeneration. As a consequence, most knee OA patients will suffer from severe pain toward disability and ultimately require total knee replacement (TKR). ⁷ The increasing rates of TKR highlight the importance of not only developing more effective disease-modifying treatments but also identifying potential biomarkers to aid with earlier diagnosis to prolong the period before patients experience significant disability. Given that synovial inflammation is directly linked to clinical symptoms and also reflects joint destruction in knee OA, it seems likely that treating key aspects of synovial inflammation hold great promise not only for painlessness but also for prevention of structural modification in the patients. For this reason, the ability of inflammatory molecules to participate in synovitis process would be of great interest to many researchers for exploring clinical utility of those mediators as biomarkers for knee OA.

Of various cytokines produced in the inflamed synovium, interleukin-34 (IL-34), a newly discovered inflammatory cytokine, is becoming increasingly recognized as a possible mediator for synovitis in knee OA. As an alternative ligand for colony-stimulating factor-1 receptor (CSF-1R), IL-34 shares functional similarities with CSF-1 (also known as macrophage-colony-stimulating factor, M-CSF) that regulates a variety of biological effects on several cells—especially osteoclasts.^8-10 With regard to its biological function in osteoclasts, IL-34 has been shown to stimulate the formation and activation of osteoclasts, which in turn magnifies osteoclasts-resorbing activity, thereby establishing the pathological role of IL-34 in bone resorption. ¹¹ Apart from its action in osteoclastogenesis, IL-34 has been reportedly implicated in synovial inflammation through enhancing production of inflammatory mediators,^12,13 in which altered IL-34 mRNA expression is regulated by pro-inflammatory cytokines responsible for cartilage degradation. ¹² Notably, emerging evidence has accumulated an increase in the systemic and local production of IL-34 in patients with arthritis, particularly in rheumatoid arthritis (RA) patients, and its systemic levels were positively associated with the disease severity. ¹⁴ Collectively, the important roles of IL-34 in osteoclastogenesis and synovial inflammation have made it a possible biomarker for joint-related diseases, including knee OA.

To the best of our knowledge, although the possible role of IL-34 involved in bone erosion and synovial inflammation and its levels in the systemic and local joint environments in patients with inflammatory arthritis have been somewhat understood and partially explored, the circulating and synovial fluid IL-34 of patients with primary knee OA and its significant involvement in the OA patients have received little attention. Accordingly, the objectives of this study were to investigate IL-34 levels in the circulation and joint fluid of knee OA patients compared with plasma healthy controls and to determine whether its levels were associated with radiographic severity of the patients. In addition, both mRNA and protein expressions of IL-34 were examined in the inflamed and noninflamed synovial tissues obtained from knee OA patients.

Materials and Methods

The study protocol conducted in accordance with the guidelines of the Declaration of Helsinki was approved by the Institutional Review Board on Human Research of the Faculty of Medicine, Chulalongkorn University (IRB No. 533/54) and the Faculty of Dentistry/Faculty of Pharmacy, Mahidol University (IRB No. 2018/072.1812). A written informed consent was acquired from all participants prior to their enrolment in the study.

Study Subjects

Ninety-six patients with primary knee OA who met the criteria of the American College of Rheumatology for knee OA and 72 healthy controls without clinical or radiological evidence of OA were included in this case-control study. All knee OA patients were scheduled to undergo either therapeutic arthroscopy or TKR. Of 96 knee OA patients, 21 underwent consecutive unilateral TKR, and 75 received therapeutic arthroscopy. The participants who had underlying diseases such as diabetes mellitus, advanced liver or renal diseases, histories of either long-term steroid treatment or anti-inflammatory drugs, other forms of arthritis, previous knee injury, infection, malignancy, and/or other chronic inflammatory diseases were excluded from this study, given that the aforementioned factors may influence circulating IL-34 levels. The healthy controls who attended an annual health check-up at King Chulalongkorn Memorial Hospital were recruited using a convenience sampling based on medical records and consultations, if they met the following inclusion criteria: (1) they had no clinical indicators and symptoms of arthritis or joint-related disorders and (2) they had no radiological signs of OA. Prior to inclusion within the study, all healthy volunteers underwent a knee radiograph to attest that they had no radiographic features of OA.

Knee radiograph was taken when each participant was standing on both legs with fully extended knees, and the X-ray beam was centered at the center of the joint. Assessment of radiographic severity was performed using the Kellgren-Lawrence (KL) system being a common method used to classify severity of knee OA, ¹⁵ and all preoperative radiographs were assessed in a blinded manner to the patient’s clinical and laboratory data. Depending on changes observed in standard weightbearing anteroposterior radiograph of the affected knees, the radiographic change of knee OA was categorized into 5 grades (0 to 4): grade 0 (normal findings with no X-ray changes), grade 1 (doubtful narrowing of joint space and possible osteophyte lipping), grade 2 (definite osteophytes and possible joint space narrowing), grade 3 (moderate multiple osteophytes, definite narrowing of joint space, bone sclerosis, and possible deformity of bone contour), and grade 4 (large osteophytes, marked joint space narrowing, severe sclerosis, and clear deformity of bone contour). In general, knee OA patients were defined as having radiographic knee OA of KL grade ≥2 in at least 1 knee. Healthy volunteers were defined as having neither radiographic hip OA nor knee OA, as indicated by KL grades of 0 for both hips and both knees.

Quantitation of Plasma and Synovial Fluid IL-34 levels

Venous blood samples were collected from all participants following 12-hour overnight fasting into ethylenediaminetetraacetic acid (EDTA) tube, centrifuged, and stored immediately at −80°C until utilized. Synovial fluid was aspirated approximately 1 to 2 mL from one knee of the OA patients found higher KL grade than another one using sterile needle and syringe during surgery and then collected into EDTA tube. The joint aspiration was performed by a doctor. The synovial fluid samples were subsequently centrifuged to remove cells and joint debris and kept at −80°C for further measurement. Plasma and synovial fluid IL-34 levels were quantified using enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN, USA). According to the manufacturer’s instructions, recombinant human IL-34 standards, plasma, and synovial fluid samples were pipetted into every well of a microplate, which was precoated with specific antibody to IL-34. After incubating for 2 hours at room temperature, all wells were washed completely 3 times with washing reagent. Successively, IL-34 conjugate was pipetted into every well and incubated for 2 hours at room temperature. After 3 washes, substrate solution was added into each well, and then the microplate was incubated for 20 minutes at room temperature without light. Finally, all reactions were terminated by the stop reagent, and the optical density was evaluated using automated microplate reader at 450 nm. The intensity of color derived is clearly proportionate to the quantity of IL-34 in the samples. A standard optical density–concentration curve was depicted for the determination of IL-34 value ranging from 0.0625 to 4 ng/mL. The intra- and interassay coefficients of variation (CVs) were 1.8% to 7.3% and 4.1% to 6.0%, respectively. The detection limit of this assay was 1.78 pg/mL. For the technical validity of measurements, plasma and synovial fluid samples were analyzed for human IL-34 at a 10-fold dilution.

Determination of IL-34 mRNA Expression

Synovial biopsies from suprapatellar pouch of the affected joint of 30 knee OA patients were harvested surgically at the time of arthroplasty, in which 1 (3.3%) was defined as KL grade 2, 9 (30.0%) were defined as KL grade 3, and 20 (66.7%) were defined as KL grade 4. Of these, 15 synovial tissues were identified as the inflamed synovium, based on histological findings assessed by a pathologist who was blinded to clinical status and diagnosis of the patients. Relative IL-34 mRNA expression was determined in the inflamed (n = 15) and noninflamed synovial tissues (n = 15) obtained from knee OA patients who underwent TKR using quantitative real-time polymerase chain reaction (PCR). Total RNA was isolated using RNeasy Mini Kit (Qiagen, Hilden, Germany), with cDNA reverse transcribed using TaqMan Reverse Transcription Reagents (Applied Biosystems, Inc., Foster City, CA, USA). Real-time PCR was performed using QPCR Green Master Mix HRox (biotechrabbit GmbH, Hennigsdorf, Germany) on StepOnePlus Real-Time PCR System (Applied Biosystems, Inc., Foster City, CA, USA). Primers used for IL-34 and glyceraldehyde 3-phosphate dehydrogenase (GADPH) amplification were, as follows: IL-34 forward primer 5′- GTGCTTAGGCCTCTGTGGAC-3′, IL-34 reverse primer 5′-GCCAAGGAAGATCCCAAGATA-3′; GADPH forward primer 5′-GTGAAGGTCGGAGTCAACGG-3′, and GADPH reverse primer 5′-TCAATGAAGGGGTCATTGATGG-3′. Real-time PCR conditions were performed, as follows: (initial step) 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds, and then 56°C for 1 minute. Relative IL-34 mRNA expression normalized to GADPH as an internal control was determined using 2^−∆∆Ct method.

Determination of IL-34 Protein Expression

Localization of IL-34 protein expression in the synovial biopsies of knee OA patients was examined using immunohistochemical analysis. Tissue specimens were paraffin-embedded and subsequently sectioned, according to standard protocols. Routine staining with hematoxylin and eosin (H&E) and immunohistochemistry staining with antibodies was performed to detect IL-34 protein expression (Abcam, Cambridge, MA, USA). A standard immunohistochemical technique was performed using a Ventana Benchmark XT autostainer (Ventana Medical Systems Inc., Tucson, AZ, USA). Briefly, tissue sections were deparaffinized and rehydrated. Endogenous peroxidase activity was blocked by 0.3% hydrogen peroxide for 10 minutes. Following heat-induced antigen retrieval in 10 mmol/L citrate buffer (pH 6.0) for 5 minutes, the slides were incubated in pepsin for 7 minutes and subsequently incubated with 1:500 diluted primary antibodies for 2 hours. Afterward, the sections were stained with the secondary antibody conjugated to streptavidin/horseradish peroxidase for 45 minutes at room temperature. Reaction products were visualized using 3,3-diaminobenzidine tetrahydrochloride (Sigma, St. Louis, MO, USA), and the sections were counterstained with hematoxylin. The classification of synovial inflammation was evaluated using H&E staining with pathologist’s assessment, according to the following relevant morphological alterations: hyperplasia of synovial lining cell layer, activation of synovial stroma, and infiltration of inflammatory cells.

Statistical Analysis

Statistical analyses were accomplished with the Statistical Package for Social Sciences (v. 22.0, IBM Corp., Armonk, NY, USA). Demographic and clinical characteristics between groups were evaluated using chi-square tests and Student t test where appropriate. The Kolmogorov-Smirnov test and quantile-quantile plot were used to determine whether IL-34 mRNA and protein levels were normally distributed, in which IL-34 protein levels were normally distributed, whereas IL-34 mRNA expression exhibited skewed distribution. Comparisons between means were evaluated by Student t test (for 2 groups) or 1-way analysis of variance (ANOVA, >2 groups) with a Tukey post hoc test, while Mann-Whitney U test and Kruskal-Wallis H test were utilized for comparison of abnormally distributed continuous variables. Correlations between IL-34 levels and clinical parameters were assessed using Spearman’s rank correlation coefficient test (r). Multivariate logistic regression models were employed to determine the roles of confounding factors and to identify risk factors for advanced knee OA. Receiver operating characteristic (ROC) curve and the area under the ROC curve (AUC) were constructed to estimate the specificity and sensitivity of predicting knee OA severity using IL-34 values. Data are represented as mean ± standard deviation (SD). Statistical significance for differences and correlations was set at P < 0.05.

Results

Baseline Characteristics of Study Participants

The detailed demographic characteristics of 96 knee OA patients and 72 healthy controls are summarized in Table 1 . Mean age, gender ratio, and body mass index (BMI) in knee OA patients and healthy controls were not significantly different. On the basis of the radiographic classification, knee OA patients were classified into 3 subgroups corresponding to KL grade in the one found higher on comparison between both knees (KL grade 2: 31 [32.3%]; KL grade 3: 29 [30.2%]; KL grade 4: 36 [37.5%]). There were no significant differences in age, gender ratio, and BMI values among knee OA subgroups. Of 96 knee OA patients, 78 (81.2%) were bilateral knee OA patients, and 18 (18.8%) were unilateral knee OA patients. Considering KL grade in the contralateral joint of bilateral knee OA patients, 23 (29.5%) were defined as KL grade 2, 27 (34.6%) were defined as KL grade 3, and 28 (35.9%) were defined as KL grade 4.

Table 1.

Baseline Characteristics of Healthy Controls and Knee OA Patients among KL Subgroups.

Variables	Controls	Knee OA Patients				P
		Total	KL Grade 2	KL Grade 3	KL Grade 4
Number	72	96	31	29	36
Age (years)	69.2 ± 6.1	70.9 ± 7.9	70.6 ± 8.4	70.2 ± 8.6	71.8 ± 7.2	0.69
Gender (female/male)	50/22	79/17	22/9	26/3	31/5	0.13
BMI (kg/m2)	26.4 ± 3.6	26.7 ± 3.6	26.6 ± 3.6	26.5 ± 4.2	26.9 ± 3.6	0.89

BMI = body mass index; KL = Kellgren and Lawrence; OA = osteoarthritis.

IL-34 Levels in Knee OA Patients among Different Subgroups

As depicted in Fig. 1A , mean plasma IL-34 levels were significantly greater in knee OA subjects than that in healthy controls (P < 0.001). Although mean IL-34 levels were higher in synovial fluid than that in paired plasma of knee OA patients, this difference was not statistically significant. Given that elderly women had a high prevalence of knee OA and may be possible confounding factors affecting the result, multivariate logistic regression analysis was further performed. After adjusting for age, gender, and BMI, plasma IL-34 levels remained significantly higher in knee OA patients than that in healthy controls (odds ratio [OR] = 1.001, 95% CI = 1.000-1.001, P = 0.002).

Figure 1.

Plasma and synovial fluid interleukin-34 (IL-34) levels in subjects among different groups. (A) Plasma and joint fluid IL-34 levels in healthy controls (3811.3 ± 693.7 pg/mL for plasma IL-34) and knee osteoarthritis (OA) patients (4405.3 ± 1260.5 pg/mL for plasma IL-34, 4504.8 ± 1266.6 pg/mL for synovial fluid IL-34). (B) Plasma IL-34 in knee OA subgroups including Kellgren-Lawrence (KL) 2 (3391.0 ± 970.0 pg/mL), KL 3 (4345.2 ± 834.0 pg/mL), and KL 4 (5327.2 ± 1078.0 pg/mL) according to radiographic severity. (C) Synovial fluid IL-34 in knee OA subgroups including KL 2 (3729.4 ± 709.1 pg/mL), KL 3 (4417.6 ± 918.2 pg/mL), and KL 4 (5242.8 ± 1466.4 pg/mL) according to radiographic severity. (D) Direct link between plasma and synovial IL-34 levels in knee OA patients.

In analyses stratified by knee OA severity with regard to KL grade, the patients with higher radiographic severity exhibited remarkably more pronounced IL-34 levels in both plasma and joint fluid (P < 0.001) ( Fig. 1B and C ). In this regard, compared with knee OA patients having KL grade 2 and 3, plasma IL-34 levels in those with KL grade 4 were considerably elevated (P < 0.001, P < 0.001, respectively) ( Fig. 1B ). In conjunction with these results, knee OA patients with KL grade 4 presented markedly higher synovial fluid IL-34 levels than those with KL grades 2 and 3 (P < 0.001, P = 0.004, respectively) ( Fig. 1C ). Multivariate logistic regression analysis with adjustments for age, gender, BMI, and contralateral KL grade demonstrated a significant increase in plasma IL-34 levels in the patients with KL grade 4 when compared with those with KL grade 2 and 3 (OR = 1.002, 95% CI = 1.001-1.003, P < 0.001; OR = 1.001, 95% CI = 1.000-1.002, P = 0.002; respectively), which was in line with determination on synovial fluid IL-34 levels (OR = 1.001, 95% CI = 1.001-1.002, P < 0.001; OR = 1.001, 95% CI = 1.000-1.001, P = 0.009, respectively).

To further identify possible factors associated with risk of advanced knee OA, we performed multivariate logistic regression analysis. The association between clinical variables and risk of severe knee OA is demonstrated in Table 2 . After adjusting for age, gender, BMI, plasma IL-34, and synovial fluid IL-34, both plasma and synovial fluid IL-34 levels were found to be independently associated with increased risk of advanced knee OA patients who were defined as either KL grade 3 or 4 (OR = 1.15, 95% CI = 1.06=1.25, P = 0.001; OR = 1.16, 95% CI = 1.06-1.27, P = 0.002, respectively).

Table 2.

Multivariate Regression Analysis for Identifying Risk Factors of Advanced Knee OA. ^a .

Variables	Knee OA Patients with and without Advanced Stage
	Adjusted Odds Ratio (95% CI)	P b
Age (years)	1.06 (0.94-1.19)	0.34
Gender (female/male)	1.50 (0.13-17.08)	0.74
BMI (kg/m2)	1.13 (0.91-1.39)	0.27
Plasma IL-34 (pg/mL)	1.15 (1.06-1.25)	0.001
Synovial IL-34 (pg/mL)	1.16 (1.06-1.27)	0.002

BMI = body mass index; IL-34 = interleukin-34; OA = osteoarthritis.

^aOdds rratio was adjusted for age, gender, BMI, plasma IL-34, and synovial IL-34.

^bP values in boldface are statistically significant.

Correlations between Plasma and Synovial Fluid IL-34 and Knee OA Severity

Given plasma and synovial fluid IL-34 levels as independent risk factors of advanced knee OA, we determined the relationships between plasma and synovial fluid IL-34 levels with the radiographic severity of knee OA. Plasma IL-34 levels were directly correlated with radiographic severity (r = 0.64, P < 0.001). Similarly, synovial fluid IL-34 levels were positively associated with knee OA severity (r = 0.50, P < 0.001). Further analysis revealed a positive association between plasma IL-34 levels and synovial fluid IL-34 levels in knee OA patients (r = 0.64, P < 0.001) ( Fig. 1D ).

Plasma and Synovial Fluid IL-34 Levels as Possible Biomarkers for Knee OA Severity

To explore whether plasma and synovial fluid IL-34 levels allow distinguishing knee OA patients with advanced stage (KL grade 3-4) from those with early-stage (KL grade 2), we calculated the AUC of the ROC curve, which was constructed using IL-34 values. Diagnostic value of plasma and synovial fluid IL-34 levels as biomarkers for knee OA severity is depicted in Fig. 2 . The ROC curve analysis illustrated that the optimal cutoff value of plasma IL-34 as a useful biomarker for advanced knee OA was projected to be 3750.0 pg/mL, which yielded a sensitivity of 83.1%, a specificity of 74.2%, and an AUC of 0.85 (95% CI = 0.78-0.93; P < 0.001). For synovial fluid IL-34 analysis, the optimal cutoff value of synovial fluid IL-34 was defined at 3950.0 pg/mL, and the AUC was 0.75 (95% CI = 0.65-0.85; P < 0.001). Sensitivity and specificity of synovial fluid IL-34 as a biomarker for advanced knee OA were 70.8% and 71.0%, respectively.

Figure 2.

Receiver operating characteristic curve showing diagnostic value of plasma and synovial fluid interleukin-34 (IL-34) as biomarkers for distinguishing knee osteoarthritis (OA) patients with advanced stage from those with early stage.

Expressions of IL-34 mRNA and Protein in the Knee OA Synovium

Owing to increased plasma and synovial fluid IL-34 levels in knee OA patients—especially those with advanced stage, we investigated relative IL-34 mRNA expression in the inflamed synovial tissues (n = 15) and the noninflamed synovial tissues (n = 15) of knee OA patients using quantitative real-time PCR. Compared with the noninflamed synovial tissues, relative IL-34 mRNA expression was significantly increased in the inflamed synovial tissues of knee OA patients (P < 0.001) ( Fig. 3A ). In multivariate logistic regression analysis with adjustments for age, gender, BMI, relative IL-34 mRNA expression in the inflamed synovial tissues of knee OA was found to be significantly higher than that in the noninflamed synovial tissues of knee OA (OR = 6.22, 95% CI = 1.63-27.73, P = 0.007).

Figure 3.

Relative IL-34 mRNA expression in knee osteoarthritis (OA) synovium and its protein levels in knee OA patients with and without synovitis. (A) IL-34 mRNA expression in noninflamed and inflamed synovial tissues (1.3 ± 0.8, 2.9 ± 1.6, respectively). (B) Plasma IL-34 levels in knee OA patients with and without synovitis (4643.2 ± 1585.1 pg/mL, 2607.2 ± 380.3 pg/mL, respectively). (C) Synovial fluid IL-34 levels in knee OA patients with and without synovitis (4793.4 ± 1572.1 pg/mL, 3163.4 ± 605.3 pg/mL, respectively).

Apart from IL-34 mRNA expression in 30 synovial tissues of knee OA, IL-34 protein levels in available plasma and synovial fluid samples from 21 knee OA patients were measured, in which 15 were identified as the patients with synovitis, and 6 were defined as the patients without synovitis with regard to histological findings with H&E staining. As depicted in Fig. 3B and C , the patients with synovitis had significantly greater plasma IL-34 levels than those without synovitis (P = 0.006), correspondent with analysis of synovial fluid IL-34 levels (P = 0.025). After adjusting for age, gender, BMI as confounding factors, multivariate logistic regression analysis demonstrated a significant increase in plasma IL-34 levels in the patients with synovitis, compared with those without synovitis (OR = 1.003, 95% CI = 1.000-1.006, P = 0.037) and no significant difference in synovial fluid IL-34 levels between the patients with and without synovitis (OR = 1.01, 95% CI = 0.99-1.02, P = 0.073).

Aside from its mRNA expression, we additionally determined localization of IL-34 protein expression in the inflamed synovium compared with the noninflamed synovium. Histolopathogical findings of knee OA synovium with and without synovitis are represented in Fig. 4A-E . In histopathology of knee OA synovitis ( Fig. 4B ), H&E staining depicted hypertrophy of the synovial lining layer ( Fig. 4C ), presence of synovial stoma ( Fig. 4D ), and cellular infiltration with lymphocytes ( Fig. 4E ), contrary to the noninflamed synovium demonstrating absences of enlargement of the lining layer, synovial stoma, and inflammatory infiltration ( Fig. 4A ). For immunohistochemistry staining analysis demonstrated in Fig. 5A-E , IL-34 protein was expressed throughout the synovial lining layer by synoviocytes (arrows) and inflammatory cells (dotted arrows) ( Fig. 5C ) and the sublining layer by endothelial cells (arrowheads) and fibroblasts (white arrows) at the inflamed synovium ( Fig. 5B-E ). In contrast to the knee OA inflamed synovium, IL-34 protein expression was scarcely evident in the noninflamed synovium of knee OA, demonstrated as faint cytoplasmic staining ( Fig. 5A ).

Figure 4.

Histopathological staining of knee osteoarthritis (OA) synovium. (A) No synovitis. (B) Synovitis. (C) Hypertrophy of the synovial lining layer. (D) Increased density of the synovial stroma. (E) Inflammatory lymphocytic infiltration.

Figure 5.

Immunohistochemical staining for interleukin-34 (IL-34) protein expression. Specific staining of IL-34 protein is present by brown coloration. Expression of IL-34 was observed in the synovial lining cell layer, predominantly in synoviocytes (arrows) and inflammatory cells (dotted arrows) and the synovial sublining cell layer, especially in endothelial cells (arrowheads) and fibroblasts (white arrows). (A) IL-34 protein expression in knee osteoarthritis (OA) synovium without synovitis. (B) Knee OA synovium with synovitis. (C) The synovial lining layer of knee OA synovitis. (D) The synovial sublining layer of knee OA synovitis. (E) Inflammatory infiltration of knee OA synovitis.

Discussion

Given that IL-34 is considered to be a novel inflammatory cytokine functionally implicated in synovial inflammation in arthritis-related immune disease, it is not surprising that IL-34 may have an immense potential to be a biologically active mediator of synovial inflammation-induced joint degeneration in knee OA. As to its primary action as an alternative ligand for CSF-1R, IL-34 can substitute CSF-1 to support receptor activator of nuclear factor-κB ligand (RANKL)-induced the differentiation and activation of osteoclasts contributing to bone erosion.^11,17 With such its potent effect on osteoclastogenesis, IL-34 has been recognized as a downstream effector of pro-inflammatory cytokines responsible for synovial inflammation, which is mediated through the activation of nuclear factor kappa B and c-Jun N-terminal kinase signaling pathways.^12,18 It has been well-recognized that pro-inflammatory cytokines, including IL-1β and tumor necrosis factor-α (TNF-α) have a chief action in promoting synovial inflammation in knee OA by heightening the release of inflammatory mediators, matrix metalloproteases, and chemokines from fibroblast-like synoviocytes into synovial fluid, which in turn damage surrounding tissues of the joint—predominantly the synovial membrane.^2,16 In addition to the synovium, inflammatory cytokines have been reportedly released from the IFP. ⁵ The synovium/IFP complex has gained increasing attention as a potential therapeutic target in knee OA patients. The above-mentioned findings give emphasis to the possible involvement of IL-34 in synovial inflammation of knee OA and support our own findings, in which significantly increased levels of IL-34 were observed in plasma and synovial fluid samples of knee OA patients, and its levels were positively correlated with knee OA severity. In accordance with our main results, a recent study by Wang et al. ¹⁹ denoted that synovial fluid IL-34 levels were considerably greater than that in paired serum samples of knee OA patients, and its synovial fluid levels were positively associated with radiographic severity and scores of knee pain and disability. Besides increases in the systemic and local levels of IL-34 in knee OA patients, a number of clinical studies showed that elevated levels of circulating and synovial fluid IL-34 were directly associated with clinical variables of RA patients.^20-23 Strikingly, IL-34 mRNA expression was reportedly raised in the inflamed synovial tissues of RA patients.^20,24 In parallel with this previous finding, overexpression of IL-34 protein was ubiquitously positioned throughout RA synovium with synovitis, and its expression was found to be closely correlated with severity of synovitis. ²⁰ These previous findings attest subsequent results derived from our study, which unveiled upregulation of IL-34 mRNA expression in the inflamed synovial tissues of knee OA patients compared with the noninflamed synovial tissues. In addition to altered IL-34 mRNA expression in knee OA synovitis, our additional analysis showed that IL-34 protein was expressed in the inflamed synovium of knee OA—notably in the enlarged synovial lining cell layer. All our findings led us to speculate that increases in circulating and synovial fluid IL-34 levels would reflect knee OA severity, especially inflammation of synovial membrane, and circulating IL-34 may have a diagnostic value as a noninvasive biomarker for monitoring the severity of knee OA. To address these hypotheses, we further performed ROC curve analysis showing the clinical usefulness of plasma IL-34 as a novel biomarker for discriminating knee OA patients with advanced stage from those with early stage, which yielded greater values of sensitivity and specificity than other molecules, including matrix metalloproteinase-3 and cartilage oligomeric matrix protein as previously described.^25,26 Our finding highlights its possible potential as a biomarker for monitoring the disease severity. As a diagnostic value of circulating IL-34 for monitoring poor outcomes of knee OA patients derived from ROC curve analysis showed an AUC of less than 0.90, it is important to note that a combination of circulating IL-34 and existing indicators can bring about improvements in the assessment of clinical outcomes in knee OA patients. Even though the mechanisms underlying alterations in the systemic and local production of IL-34 in knee OA still remain largely unclear, the possible reason for raised IL-34 levels in plasma and synovial fluid of knee OA patients and those with advanced stage may be attributed to compensatory mechanisms of the body that become activated in response to the imbalance between cartilage damage and subchondral bone remodeling and further synovial inflammation. The aforementioned circumstance could enhance the release of IL-34 from the local tissues like the inflamed synovium into the circulation. This assumption has been supported by our results revealing overexpression of IL-34 mRNA and protein in the inflamed synovium of knee OA patients.

In spite of significant findings presented herein, the current study inevitably had some inherent caveats, which require to be taken into account. The most notable limitation is the fact that the study is cross-sectional in design with a relatively small number of participants that prevents the determination of cause-and-effect relationships. It is recommended that multicenter prospective cohorts with larger sample sizes are needed to verify any relationships. Another caveat is the lack of patient data on physical performance tests and clinical symptoms, including knee pain and joint function. As a result, we were unable to determine the possible associations of IL-34 levels with functional impairment (Western Ontario and McMaster Universities Osteoarthritis, WOMAC score) and severity of knee pain (visual analogue scale) in knee OA patients. In this context, additional studies are warranted to clarify these associations. Unfortunately, due to ethical considerations, direct comparisons between synovial fluid IL-34 of controls and that of knee OA patients were unachievable as a consequence of being unable to collect joint fluid specimens from healthy volunteers. In future studies, this may be overcome by the use of nonarthritic knee surgery patients as controls.

To sum up, the present study provides additional evidence supporting an increase in circulating and synovial fluid IL-34 levels in knee OA patients, particularly in the patients with advanced stage. Interestingly, there was a significantly positive correlation between plasma and synovial fluid IL-34. Furthermore, both plasma and synovial fluid IL-34 levels were shown to be positively correlated with radiographic severity of knee OA patients. Subsequent analysis demonstrated overexpression of IL-34 mRNA and protein in the inflamed synovial tissues of knee OA. Collectively, IL-34 appears to have potential as a biochemical parameter reflecting knee OA severity. Further understanding of the exact role of IL-34 in the pathogenesis of knee OA will provide new therapeutic approaches to the treatment of knee OA.