Background
Rheumatoid arthritis (RA) is a chronic inflammatory condition that can cause joint degeneration over time [
1]. In RA joints, a variety of cell types, including innate immune cells, adaptive immune cells, endothelial cells, and fibroblast-like synoviocytes (FLSs), are activated. In particular, synoviocytes play an important role in the invasive pannus and directly contribute to chronic inflammation and cartilage degradation [
1,
2]. They are capable of producing matrix-degrading enzymes and cytokines, such as IL-6 and IL-8, as well as angiogenic factors. Furthermore, fibroblast-like synoviocytes from inflamed joints can grow abnormally, migrate to the local environment, and exhibit tumor-like features [
3,
4].
Synovial tissue inflammation is known to contribute to RA development. It exists in all stages of RA, even in its early stages. Numerous proinflammatory cytokines, including tumor necrosis factor (TNF), IL-6, IL-8, IL-17, and CCL2, contribute to the progression of RA [
5]. An increase in joint infiltration by immune cells, specifically macrophages, has been closely linked to elevated CCL2 levels in RA [
6]. Synovial inflammation also promotes synovial angiogenesis in the synovium, which, in turn, accelerates inflammation [
3,
7]. Therefore, controlling the inflammatory response is a promising strategy for the treatment of RA.
Human high-temperature requirement serine protease A (HtrA2), also known as Omi, is a mitochondrial serine protease. It is an apoptosis-inducing protein that is released from the mitochondrial intermembrane space into the cytosol following an apoptotic stimulus [
8]. HtrA2 plays a role in the progression of neurodegenerative diseases, prostate cancer, and hepatocellular carcinoma [
8,
9]. HtrA1, a paralog of HtrA2, is also involved in the development of various skeletal diseases including RA [
10,
11]. Of note, HtrA1 and HtrA2 of the HtrA2 family of serine proteases appear to share substrate specificity. These studies suggest that HtrA2 modulates the progression of RA [
10,
12]; however, the molecular mechanisms through which HtrA2 controls inflammation and the immune response are unclear. Moreover, most studies on its disease association have been carried out using the proapoptotic mitochondrial serine protease. In the present study, we demonstrate that HtrA2 mediates the pathogenesis of inflammatory arthritis. HtrA2 was observed to be abundant in RA synovium samples and synovial fluids from patients. Synovial HtrA2 levels were directly associated with synovial IL-6, IL-8, and CCL2 levels (representative proinflammatory cytokines/chemokines). In addition, ER stress stimuli such as tunicamycin and thapsigargin released HtrA2 in cultured synoviocytes. For the first time, we demonstrated that HtrA2 is secreted extracellularly during ER stress-induced apoptosis. Furthermore, siRNA-mediated downregulation of HtrA2 transcripts reduced the production of inflammatory cytokines and chemokines (IL-6, IL-8, and CCL2) in RA FLSs while eradicating IL1β-, TNFα-, or LPS-triggered cytokine production. Overall, our data show that HtrA2 is a novel inflammatory mediator. Collectively, our finding provides new insights into the use of HtrA2 as a novel biomarker of RA and the design of next-generation therapeutic strategies for RA, as the production of other inflammatory mediators is reduced by modulating the amount of HtrA2.
Methods
Study population and sampling of SF and serum
Individuals with RA who met the 2010 ACR/EULAR categorization criteria [
13] and osteoarthritis (OA) controls were enrolled between August 2015 and December 2018. SF samples were collected from individuals with RA (
n = 72) and an OA control group (
n = 61), who were subjected to arthrocentesis for swollen joints. SFs were centrifuged at 6000
g for 15 min after they were aspirated from affected joints. Supernatants were stored at −80 °C.
Assessment of synovitis severity by ultrasonography
Sonographic evaluations of joints including grayscale US (GSUS) and power Doppler US (PDUS) were conducted as previously reported [
14]. In summary, GSUS defined the degree of synovial hypertrophy as follows: grade 0 = no synovial hypertrophy; grade 1 = minimal synovial hypertrophy; grade 2 = moderate synovial hypertrophy; and grade 3 = severe synovial hypertrophy. PDUS scores were evaluated by the degree of vascularity within synovium of joints as follows: grade 0 = absence of Doppler signal; grade 1 = minimal Doppler signal; grade 2 = moderate Doppler signal (≥grade 1 but < 50% of Doppler signals in the total background); grade 3 = high Doppler signal (> 50% of Doppler signals in the total background). Individuals were characterized as having active synovitis if GSUS grade or more ≥2 or if PDUS grade was 1 or more ≥1.
Isolation and culture of fibroblast-like synoviocytes (FLS)
Fibroblast-like synoviocytes (FLSs) were isolated from synovial tissues of individuals with rheumatoid arthritis (RA) and osteoarthritis (OA) who had complete joint replacement surgery. FLSs were extracted from synovial tissues as previously reported [
15,
16]. FLSs from passages 4–8 were seeded into 24-well plates at a density of 2×10
4 cells/well in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% Fetal bovine serum (FBS), penicillin, streptomycin, and amphotericin B.
Downregulation of HtrA2 transcripts
To downregulate HtrA2 transcripts, Lipofectamine 3000 (Invitrogen) was used to transfect RA FLSs with HtrA2 siRNA (Santa Cruz Biotechnology, Inc.). Control siRNAs were also obtained from Santa Cruz Biotechnology, Inc. HtrA2 mRNA and protein expression levels in FLSs were evaluated by RT-PCR including Western blot analysis relying on anti-HtrA2 monoclonal antibody (Abcam), respectively. To detect HtrA2 mRNA, RT-PCR was conducted with the following HtrA2-specific primers: sense, 5′-GCACTGCAGAACACGATCAC-3; and antisense, 5′-GGGACCTCCAGAGTTTCCAA-3′. GAPDH mRNA expression was used as an internal control.
Enzyme-linked immunosorbent assay (ELISA)
ELISA kits for human IL-6 (R&D Systems), IL-8 (R&D Systems), CCL2 (R&D Systems), TNFα (R&D Systems), and HtrA2 (R&D System) were used to measure cytokine concentration in culture supernatants and SFs.
Cell viability: MTT assay
Synoviocyte viability was assessed by an MTT assay as previously reported [
15,
16].
Immunohistochemical staining
Formalin-fixed paraffin-embedded synovial tissue specimens sectioned at 5 μm in thickness were obtained from four RA patients and four OA patients. The tissue was deparaffinized in xylene, rehydrated in a gradient series of ethanol, microwaved, and treated with 3% hydrogen peroxide to inhibit endogenous peroxidase activity. Following blocking nonspecific binding using 10% normal horse serum for 1 h at ambient temperature, the slides were treated with rabbit anti-HtrA2 Ab (1:100; Abcam) overnight at 4°C. Isotype control antibody was applied as a negative control. After washing, the slides were incubated for 30 min at room temperature with goat anti-rabbit IgG secondary Ab (ImmPRESS HRPTM REAGENT KIT; Vector Laboratories). 3,3′-Diaminobenzidine tetrahydrochloride was used to detect positive cells.
Immunofluorescence staining
A 4% paraformaldehyde solution was used to fix RA-FLSs for 20 min, then permeabilized using 0.1% Triton X-100/PBS for 15 min at ambient temperature. Following blocking for 1 h with a 10% solution of normal donkey serum, slides were stained with Abs to HtrA2 (1:100; Abcam) and Tom20 (1:100; Santa Cruz Biotechnology) for 2 h at ambient temperature. Each slide was washed three times with PBS, then incubated with an Alexa Fluor 488-tagged anti-rabbit IgG Ab (1:1000; Abcam) or Alexa Fluor 594-tagged anti-mouse IgG Ab (1:1000; Abcam) for 2 h at ambient temperature. DAPI was used to stain the nuclei and the glass slides were mounted with Vectashield Antifade Mounting Medium (Vector laboratories). CellLight Reagents BacMam 2.0 was used to stain intracellular organelles such as the mitochondria, and lysosomes, according to the manufacturer’s instructions. A confocal microscope was used to examine the stained cells (LSM 800; Cari Zeiss, Germany). The raw and deconvolved images were analyzed quantitatively using Imaris x64 v7.2 (Oxford instruments) and Zeiss ZEN software.
Western blot analysis
After lysing RA-FLS and OA-FLS samples in lysis buffer, insoluble material was removed by centrifugation for 20 min (14,000 rpm at 4°C). The BCA protein assay (Thermo Fisher Scientific) was used to estimate the protein concentration. SDS-PAGE was used to separate the proteins, which were then transferred to polyvinylidene fluoride membranes. HtrA2 (1:2000; Abcam) and the membranes were treated with β-actin antibodies (1:1000; Santa Cruz Biotechnology). After washing and incubation with secondary antibody, an enhanced chemiluminescent approach was used to visualize the protein bands.
Statistical analysis
The data are presented as the mean ± standard error of the mean (s.e.m.). The Mann-Whitney U test, Wilcoxon matched pairs test, and one-way (or two-way) ANOVA test were applied. The Spearman correlation test was used to examine the relationships between cytokines and HtrA2 levels in RA SFs. For statistical evaluations, paired or unpaired t-test was utilized, as specified in each group. P-values less than 0.05 were considered statistically significant. GraphPad Prism software v9 was used for all statistical analyses.
Discussion
HtrA2 is localized to the mitochondrial intermembrane space and influences the physiology of mitochondrial homeostasis [
8,
9,
20]. Its aberrant expression has been linked to a variety of illnesses, including ovarian cancer, endometrial cancer, and neurological disorders [
21‐
24]. In the present study, we demonstrated that HtrA2 is expressed in RA synovium and its expression is increased in synoviocytes during proinflammatory conditions. HtrA2 levels were markedly higher in the fluids of RA patients compared with that in the OA controls. To determine the relationship between HtrA2 expression and the severity of RA further, we examined its correlations with clinical variables in RA patients.
Previous research has found that HtrA2 contributes to the pathogenesis of autoimmune arthritis by regulating Th17 cells [
7]. These studies demonstrate that HtrA2 can regulate RA in part through the Th17 cell differentiation of STAT3 [
7]. In one study, UCF-101 (5-[5-(2-nitrophenyl) furfuryl iodine]-1,3-diphenyl-2-thiobarbituric acid), which is an inhibitor of HtrA2, seems to relieve pulmonary inflammation by reducing the generation of inflammatory cytokines [
25]. HtrA2 deficiency has recently been shown to reduce the production of proinflammatory cytokines in BMDMs triggered by LPS or CpG, which suggests that HtrA2 is an immune response regulator [
26]. In the present study, we clearly demonstrated that HtrA2 was a critical regulator of the production of inflammatory cytokines through HtrA2 knockdown. In particular, IL1β-, TNFα-, or LPS-induced increases of IL-6, IL-8, and CCL2 were completely blocked by HtrA2 siRNA. RA-FLSs actively participate in chronic inflammation by secreting inflammatory cytokines including IL-8 and IL-6 [
3,
4]. Furthermore, CCL2 levels were significantly upregulated in RA patients [
6,
27]. In the RA synovium, activated synoviocytes are responsible for the synthesis of CCL2, which exacerbates and sustains inflammation by recruiting inflammatory immune cells, primarily macrophages and monocytes [
6,
28‐
30]. In this regard, anti-inflammatory agents targeting RA-FLSs may exhibit a therapeutic benefit. The present study showed that HtrA2 expression was associated with increases of proinflammatory cytokine production in synoviocytes.
We observed the release of HtrA2 from mitochondria into the culture medium following treatment with an ER stress inducer. Several scientists have postulated that during apoptosis, cytochrome c is released from the mitochondria into the cytosol and ultimately into the extracellular space [
31‐
33]; however, the release of HtrA2 into the extracellular space during apoptosis has yet to be established [
19]. In the present study, we demonstrated that HtrA2 release from RA FLSs into the extracellular environment was implicated in ER-stress-induced apoptosis. Previous studies have indicated that RA joints are subject to ER stress and that ER stress-related gene signatures are expressed in RA synovial cells [
15,
16]. We hypothesize that chronic exposure of FLSs to ER stress within joints induces apoptosis, thereby releasing HtrA2.
HtrA2 is a potential biomarker for mitochondrial-induced apoptosis in the joint. However, its therapeutic utility is speculative, and further research is needed to fully grasp its role. Furthermore, there is no evidence that HtrA2 is an apoptotic marker; it might just be a marker of severe necrosis and cell lysis. Finally, our findings should be validated by larger patient population studies including further exploration into the physiology of HtrA2. More studies are necessary to determine whether HtrA2 can attach to a specific receptor or transport system at the plasma membrane and whether extracellular HtrA2 activates proapoptotic signaling pathways.
Overall, we demonstrated that HtrA2, beyond its classical role as a serine protease in the mitochondria, can acquire a new function as an inflammation regulator, exhibiting functional diversity with mitochondrial proteins. Our findings provide insight into the pathogenic mechanism of mitochondrial protein and highlight the significance of HtrA2 as a possible therapeutic target, as it is a frequent trigger of the inflammatory process.
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