CD5L induces inflammation and survival in RA-FLS through ERK1/2 MAPK pathway

Abstract

Objective

To explore the effect of CD5-like molecule (CD5L) on rheumatoid arthritis (RA) fibroblast-like synoviocytes (RA-FLS) and the relative molecular mechanism of CD5L in it.

Methods

Recombinant protein CD5L was used to stimulate the cultured RA-FLS cells. The inflammation-related cytokines were determined by real time-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). The signal molecules and apoptosis-related molecules were detected by western blot assay (WB), and cell counting kit-8 (CCK-8) was used to detect the proliferation.

Results

CD5L can increase the production of IL-6, IL-8, and TNF-α and this effect can be inhibited by signal pathway inhibitor. At the same time, CD5L activated ERK1/2 MAPK signal, inhibitor treatment can weaken the intensity of phosphorylation. In addition, CD5L can enhance the proliferation ability of RA-FLS.

Conclusion

CD5L induces the production of inflammatory cytokines in RA-FLS through the ERK1/2 MAPK pathway and increases cell survival.

Keywords:

• CD5L
• rheumatoid arthritis
• inflammatory factors
• signalling pathway
• proliferation

1. Introduction

Rheumatoid arthritis (RA) is a commonly inflammatory and chronic autoimmune disease [1,2]. It is characterized by symmetrical pain, swelling, stiffness, and even deformity of the joints of the whole body. Patients with rheumatoid arthritis are prone to severe infections, respiratory diseases, osteoporosis, cardiovascular diseases, and cancers, which lead to high mortality [2,3].

Macrophage apoptosis inhibitor (AIM, also known as CD5-like molecule (CD5L), Api6, Spα) is a soluble glycoprotein with a size of 40 kDa, which is mainly secreted by macrophages [4,5]. CD5L plays important roles in many diseases, such as metabolic syndrome [6], acute kidney injury [7]as well as cardiac rejection [8]. AIM levels were elevated in autoimmune patients with secondary progressive multiple sclerosis [9], rheumatoid arthritis [10], and osteoarthritis [11], and AIM can be used as a new sensitive biomarker to assist in the evaluation of disease activity.

CD5L regulates the progression of high-prevalence diseases, from infectious to sterile inflammatory driven diseases [12]. Whether CD5L plays a role in RA still lacks sufficient exploration. Although it has been reported that the expression of CD5L was increased in RA and was related to disease activity [10]. In this study, we will explore the effect of CD5L on RA FLS and the molecular mechanism of CD5L in it, providing a new perspective for targeted treatment of rheumatoid diseases.

2. Materials and methods

2.1. Materials

Recombinant human CD5L protein was purchased from R&D Systems (Minneapolis, USA). Anti-phospho-AKT, anti-CD36, and anti-CD55 primary antibody were purchased from proteintech (Chicago, USA). MEK inhibitor U0126 was purchased from Beyotime (Shanghai, China) and was diluent in DMSO. Anti-phospho-JAK, anti-phospho-p38 MAPK, anti-phospho-ERK, anti-phospho-IκB-α, anti-AKT, anti-JAK, anti-p38 MAPK, anti-IκB-α, anti-Bax, anti-Bcl-2, HRP-conjugated rabbit secondary antibody, and goat secondary antibody were purchased from Zenbio (Chengdu, China). Rabbit derived anti-ERK was purchased from Wanleibio(Shenyang, China). Alexa Fluor 555 labeled donkey anti-rabbit IgG (H + L) secondary antibody was purchased from Beyotime (Shanghai, China). The cell culture system was purchased from Gibco (Carlsbad, USA).

2.2. Methods

2.2.1. Extraction and culture of RA-FLS

All specimens were taken from the synovial tissue of patients with RA undergoing knee arthroplasty. Digested with type I collagenase (BioFroxx, Germany) into a single-cell suspension, and then cultured in DMEM/H medium containing 10% fetal bovine serum and 1% penicillin–streptomycin double-antibody in a 5% CO₂, 37 °C cell incubator for 48 h. According to the growth of the cells, the culture medium was replaced every 1 to 3 days. The cells cultured to 3–8 generations were used for subsequent experiments.

2.2.2. Immunofluorescence and confocal

To determine the expression of CD55 and CD36 on the RA-FLS cells, cells were fixed at room temperature with 4% paraformaldehyde, and then permeabilized with 0.1% Triton X-100 (vol/vol) (Beyontime, China). The cells were incubated with rabbit-derived anti-CD55 (1:100) or rabbit-derived anti-CD36 primary antibody (1:100) overnight at 4 °C. Next day, Alexa Fluor 555 labeled donkey anti-rabbit IgG(H + L) secondary antibody was incubated for 2 h in the dark at room temperature. The cell group that did not add Alexa Fluor 555(Alexa Fluor 555(–)) acts as a negative control to exclude false positive caused by cell self-fluorescence interference. The stained cells were observed under a confocal microscope (Nikon, Japan) after nuclear restaining with DAPI.

2.2.3. Western blotting experiment

Protein extraction kit (solarbio, China) was used to extract the total protein of synovial cells, subsequently the protein concentration was determined. The sample volume based on 30 μg protein per well was calculated. Before transferred to PVDF membrane, 12% polyacrylamide gel electrophoresis separation gel was used to separate proteins. The primary antibodies of anti-p-p38 (1:500), p-ERK(1:500), p-AKT (1:1000), p-JAK (1:500), p38 MAPK (1:500), p-IκB-α (1:500), ERK (1:500), AKT (1:500), JAK (1:500), IκB-α (1:500), Bax (1:1000), Bcl-2 (1:500) were incubated overnight at 4 °C. Next day, corresponding species secondary antibody (1:5000) was incubated for 1 h at room temperature. Then developed with enhancing sensitive chemiluminescence reagent (Millipore company, USA). GAPDH(1:10,000) (Zenbio, China) was used as internal reference control, and the gray values were analyzed by Image J software.

2.2.4. Enzyme-linked immunosorbent assay

The expression levels of IL-6, IL-8, and TNF-α in the cell culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA). The ELISA kits were purchased from Neobioscience (Shenzhen, China). The specific operation steps were as follows: First, established a standard curve. Subsequently, incubated for 90 min at 37 °C after adding the samples. The biotinylated antibody working solution and the enzyme-binding working solution were reacted in sequence. Stop the color development to detect the OD value of each well at 450 nm. The concentrations of the samples to be tested were calculated according to the standard curve.

2.2.5. Quantitative reverse transcription-PCR analysis

RNA was extracted using the E.Z.N.A® Total RNA Kit I (Omega, USA). The PrimeScript™ RT reagent Kit with gDNA Eraser (Takara, Japan) was used for reverse transcription of RNA to cDNA. All genes were analyzed on a CFX96 Real-Time PCR Detection System (BIO-RAD, USA). The specific primers were devised and composed by Takara company (shown in Table 1). The quantitative thermal cycling parameters were 95 °C for 30 s, followed by 39 cycles for 5 s at 95 °C and 30 s at 60 °C. The relative mRNA levels of IL-6, IL-8, TNF-α, BAX, and BCL-2 were evaluated and normalized with GAPDH. The data were analyzed by 2^–ΔΔCt method.

Table 1. Primers for reverse transcription-polymerase chain reaction.

Name of gene	Forward primer	Reverse primer	Product size (bp)
IL-6	5′-GAGTAGTGAGGAACAAGCCAGAG-3′	5′-GGTCAGGGGTGGTTATTGC-3′	104
IL-8	5′-TTGGCAGCCTTCCTGATTTC-3′	5′-GGGTGGAAAGGTTTGGAGTATG-3′	110
TNF-α	5′-GCCCATGTTGTAGCAAACCC-3′	5′ -TCTGGTAGGAGACGGCGATG-3′	226
BAX	5′-CCTCCTCTCCTACTTTGGGAC-3′	5′-GAAAAACACAGTCCAAGGCAG-3′	130
BCL2	5′-GTGGCCTTCTTTGAGTTCGG-3′	5′- GGTGCCGGTTCAGGTACTCA- 3′	112
GAPDH	5′-CACTCCTCCACCTTTGACGC-3′	5′-CTGTTGCTGTAGCCAAATTCGT-3′	98

IL-6, interleukin 6, IL-8, interleukin 8, TNF-α, tumour necrosis factor-α, BCL2, B cell lymphoma 2, BAX, BCL2-Associated X, GAPDH, glyceraldehyde-3-phosphate dehydrogenase; bp, base pair.

2.2.6. Cell proliferation assay

Cell proliferation was determined by cell counting kit 8 (Dojindo, Japan) according to the protocol of manufacture. Cells (1 × 10⁴) were plated in 96-well plates. At 24 h after administration of 500 ng/mL CD5L, 10 μL CCK-8 reagent was supplemented into each well and further incubated for 1 h at 37 °C. Plates were read on a microplate reader at a 450 nm. The OD values were exhibited to reveal the viability.

2.3. Statistical analysis

All data were analyzed using SPSS 20.0 (IBM SPSS Statistics, Chicago, IL, USA). Measurement data were expressed as mean ± standard deviation. Multiple comparisons were made by one-way analysis of variance. p < 0.05 indicated that the differences were statistically significant. GraphPad Prism 8 (San Diego, CA, USA) and Image J software (National Institutes of Health, Bethesda, MD, USA) were used for data mapping.

3. Results

3.1. CD55 and CD36 were expressed in RA-FLS cells

CD55 molecule (a specifically expressed molecule in RA FLS cells [13]) was chosen as a standard of identification, to determine the adopted cells (from RA surgical patients’ synovial tissues) were RA FLS cells. Uniform CD55 molecule was widely expressed in the RA FLS cells (Figure 1A) via images of confocal fluorescence staining. In addition, the expression of CD36 molecule in these cells was also detected (Figure 1B).

Figure 1. Expression of CD55 and CD36 in RA-FLS cells. A: CD55 molecule in RA-FLS. B: CD36 molecule in RA-FLS. The cells that don’t add Alexa Fluor 555 as a negative control to exclude false positive caused by cell self-fluorescence interference (×200 magnification).

3.2. CD5L promoted the expression of IL-6, IL-8, and TNF-α in RA FLS

To study the effects of different concentrations of CD5L on RA-FLS cells, we directly added different concentrations of recombinant CD5L protein to stimulate cells. The concentrations of IL-6, IL-8, and TNF-α increased to varying degrees after CD5L stimulation for 24 h (Figure 2A). According to the data, the most effective concentration of CD5L was 500 ng/mL. Similarly, the mRNA levels of inflammatory factors increased significantly (Figure 2B). At the same time, we also studied the effects of different time of CD5L stimulation on RA-FLS cells. The results showed that the level of inflammatory factors increased from 0 h to 12 h to 24 h, basically showing an upward trend, but after 24 h, the secretion of inflammatory factors reached saturation and basically remained unchanged or even slightly decreased (Figure 2C,D).

Figure 2. CD5L induced the expression of inflammatory factors in RA-FLS. The cells were stimulated with different concentrations of recombinant CD5L (0, 50, 100, 200, 500, 1000 ng/mL) for 24 h. (A) The levels of IL-6, IL-8, and TNF-α in cell culture supernatant were detected by ELISA. (B) mRNA levels of IL-6, IL-8, and TNF-α were detected by RT-PCR. (C) The levels of IL-6, IL-8, and TNF-α in cell culture supernatant after stimulating different time. (D) mRNA levels of IL-6, IL-8, and TNF-α after stimulating different time. The concentration of CD5L for stimulation was 500 ng/mL. Compared with the control group (0 ng/mL), ns, no statistical difference; *p < 0.05, **p < 0.01, ***p < 0.001. The measurements were repeated three times for each group of data.

3.3. CD5L upregulated the expression of ERK1/2 MAPK signaling pathway

A variety of signal pathways play an important role in RA. Through signal function transmission, it mediates the production, release, and function of downstream effector molecules. We firstly studied the mechanism of CD5L in RA. After CD5L stimulated cells for different periods of time, western blotting (WB) experiment was used to detect the expression of commonly classic signaling pathways. The results showed that the phosphorylation levels of p38 MAPK, PI3K/AKT, JAK, and IκBα signaling pathways were not statistically significant (Figure 3A). On the contrary, the ERK1/2 signaling pathway was obviously activated and showed a time-dependent expression (Figure 3B), which induced the strongest phosphorylation signal at 90 min.

Figure 3. CD5L activated ERK1/2 MAPK signaling pathway. Cells (5 × 10⁶) were challenged with CD5L protein (500 ng/mL) for different times(0, 30, 60, 90 min). The protein was extracted and the expression levels of phosphorylation signal molecules (A) p-p38 MAPK, p-AKT, p-JAK, and p-I κB-α and (B) p-ERK were detected by WB.

3.4. Effect of signaling pathway inhibitor on CD5L to produce cytokines in RA-FLS

To further confirm that CD5L upregulated IL-6, IL-8, and TNF-α expression were mediated by ERK1/2 signallng pathway. We pretreated cells with a specific signaling pathway inhibitor U0126 for 1 h and challenged them with CD5L for 24 h. Results showed that IL-8 and TNF-α protein concentrations in the (DMSO + CD5L) group were significantly different from those in the (U0126 + CD5L) group (p < 0.05) (Figure 4A). However, there was no significant change in IL-6 protein level between the two groups (p = 0.094) (Figure 4A). Interestingly, the CD5L group and the (U0126 + CD5L) group show comparable levels of IL-6, IL-8, and TNF-α mRNA levels (Figure 4B). Phosphorylated ERK expression was significantly reduced after U0126 rescue experiment (Figure 5). Apparently, CD5L promoted the production of inflammatory cytokines (at protein and mRNA levels), and the expression of IL-8 and TNF-α may be mediated by the ERK1/2 pathway, whereas IL-6 may be mediated by other unknown pathways.

Figure 4. Effect of inhibitor on CD5L-induced inflammatory-related factors in RA-FLS. The cells were pretreated with inhibitor U0126 for 1 h and cultured for 24 h with or without CD5L. The optimum concentration of U0126 determined by pre-experiment is 16 μM. (A) The expression levels of IL-6, IL-8, and TNF-α in the supernatant of cell culture were detected by ELISA. (B) mRNA levels of IL-6, IL-8, and TNF-α were detected by RT-PCR. Results of three independent replicates were expressed as mean ± standard deviation.

Figure 5. Effect of inhibitor on CD5L activation of ERK1/2 MAPK signaling pathway. Cells (5 × 10⁶) were treated with ERK1/2 inhibitor U0126 for 1 h, and then added CD5L protein for 90 min. Protein was extracted, and the expression level of phosphorylated signal molecule p-ERK1/2 MAPK was detected by WB. GAPDH was used to correct the protein content of each sample.

3.5. CD5L promoted RA-FLS survival and proliferation through ERK1/2 MAPK pathway

We continued to study the functional effects of CD5L on RA FLS. There was no significant difference in expression of apoptosis-related factor BAX after CD5L stimulation (p > 0.05) (Figure 6A,B). On the contrary, the mRNA expression of survival related factor BCL2 was increased fourfolds and protein was also expressed at a high level after CD5L addition (p = 0.001) (Figure 6A,B). Meanwhile, the addition of CD5L stimulated the proliferation of RA-FLS (p = 0.028) (Figure 6C). After U0126 treatment, the expression of BCL2 was significantly decreased in the presence or absence of CD5L. Similarly, the proliferation activity of RA-FLS in U0126 group and (U0126 + CD5L) group were lower than those in CD5L group (Figure 6C). Therefore, CD5L enhanced survival and proliferation through ERK1/2 pathway, but had no obvious effect on apoptosis.

Figure 6. Effects of CD5L on apoptosis and proliferation of RA-FLS. Cells with inhibitor U0126 were pretreated for 1 h and cultured for 24 h with or without CD5L. (A) mRNA levels of BAX and BCL2 were detected by RT-PCR; (B) BAX and BCL-2 protein levels were detected by WB. (C) CCK-8 kit was used to detect the proliferation ability of RA-FLS. The blank wells contained only culture medium, and the control wells contained cells and culture medium. The experimental results were expressed as mean ± standard deviation.

4. Discussion

RA is a chronic inflammatory disease characterized by joint synovial hyperplasia, cartilage degradation, and bone destruction caused by the secretion of a variety of inflammatory factors. IL-1, IL-6, and TNF-α are the most typical and common inflammatory factors in RA. However, the factors that induce the production of RA proinflammatory cytokines are still unknown so far. Our research group previously reported that CD5L was overexpressed in sera of RA [10] and SLE [14]. Proteomic analysis of synovial fluid from osteoarthritis patients revealed that CD5L was also upregulated [11].

In our study, we first identify that the adopted cells were RA-FLS (Figure 1A) and expressed CD36 molecule. Actually, CD36 is widely expressed in a variety of cells, such as monocyte-macrophages, adipocytes, muscle cells, and liver cells [15]. After CD5L stimulation, the mRNA and protein levels of chemokine IL-8, inflammatory cytokine IL-6, and TNF-α were dramatically upregulated (Figure 2A,B). Consistent with this result, the epicardial adipose tissue of heart failure patients treated with isoproterenol secretes CD5L, a molecule that may activate the Toll-like receptor 4/nuclear factor-κB (NF-κB) pathway on adjacent cells to produce proinflammatory cytokines [16].The loss of CD5L reduced the accumulation of proinflammatory M1 macrophages in myocardial tissue [17]. Similarly, inhibition of CD5L can prevent chronic vascular disease and alleviate dysfunction after myocardial infarction [17,18]. What’s more, the loss of CD5L reduced the inflammatory response and infarct size in the mouse model of acute myocardial infarction [18]. The concentrations of IL-6 in serum of acetaminophen (APAP)-treated CD5L^–/– mice were much lower than that of WT mice, and CD5L deficiency resulted in reduced APAP-induced liver inflammatory response, infiltration of neutrophils, and improved infiltration of monocytes [19]. Thus, these findings suggest that CD5L plays an important role in maintaining inflammation.

In this study, CD5L can activate ERK1/2 protein in RA FLS (Figure 3B). Rescuing experiments showed that treatment with U0126 (ERK1/2 signaling pathway inhibitor) attenuated the effect of CD5L treatment on the inflammatory phenotype of RA FLS (Figure 4A,B). Therefore, we hypothesize that CD5L mediated the production of inflammatory cytokines and chemokines through ERK1/2 signaling pathway. In the study by Li et al. [19], the expression of CD5L was increased in the liver of APAP-overtreated mice, and CD5L can directly activate JNK and ERK signaling pathways in liver cells. CD5L deficiency reduced the toxicity of APAP metabolism in the early stage, thereby attenuating the activation of JNK and ERK signaling pathways, and thus alleviating APAP-induced injury. As one of the classic MAPKs signaling pathways, ERK1/2 is sensitive to inflammatory factors such as mitogen and growth factors, thus regulates cytokine production through transcriptional and post-translational mechanisms, which is related to cell growth, proliferation, differentiation, and survival. Abnormal activation of ERK1/2 pathway was closely related to the activation of RA synovial cells and the pathological process of joint destruction [20,21].

Excessive proliferation of synovial cells and erosion of bone and cartilage are key factors in the occurrence and development of RA disease. Related studies [22,23] have shown that the frequency of apoptosis in synovial tissue was low, the apoptosis rate of fibroblasts in the lower lining layer was 3%, and there was almost no sign of apoptosis in the lining layer cells. These abnormalities were related to the abnormal expression of apoptosis-related genes. Although not statistically significant, the transcription and protein levels of proapoptotic molecule BAX were slightly decreased, whereas the anti-apoptotic factor BCL-2 was significantly at a higher level after CD5L stimulation (Figure 6A,B). Studies on the function of CD5L in synovial cells showed that it could stimulate the proliferation of cultured RA FLS (Figure 6C) and improve the survival rate. Chemical inhibition of ERK (U0126, 16 μM) in CD5L-treated primary RA FLS reduced survival ability as shown by decreased BCL-2 expression and increased BAX level.

There are some limitations to this study: first, although our study has observed the expression of CD36 in RA-FLS cells, it is not the only receptor for CD5L. Further studies are needed to determine whether CD36 is the receptor for CD5L in RA-FLS cells and how does it bind to CD5L and whether this binding is temporary, persistent, or endocytic. Second, more RA related pathogenic cells such as monocytes, macrophages, and Th17 cells should be included in future experiments to complete the role and mechanism of CD5L in RA. Finally, we simply study the inflammatory effects of CD5L mediated by ERK signaling pathway in RA-FLS, without specifically exploring how this pathway is conducted by various effector molecules. In the future, the intranuclear promoter and transcriptor of signal molecules can be further explored.

5. Conclusion

Our study firstly demonstrates the role of CD5L in RA. CD5L mediates the expression of cytokines IL-6, IL-8, and TNF-α in ERK1/2 pathway. Meanwhile, studies on the function of CD5L shows that CD5L upregulates the expression of prosurvival factor BCL-2, promotes the proliferation and survival of RA-FLS cells.

Ethics approval and consent to participate

All patients signed an informed consent form, and the research protocol was approved by the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University (No. 2021-477). All methods are carried out in accordance with relevant guidelines and regulations.

Author contribution

Huiqing Yang carried out the major experiments and wrote the main manuscript text. Yan Luo provided technical and academic guidance. Xiaofei Lai modified the manuscript and provided financial support. All authors reviewed the manuscript.

Acknowledgements

We thank the Clinical Laboratory of the Department of Medical Laboratory and the Medical Experimental Center of the First Affiliated Hospital of Chongqing Medical University for their equipment and technical support.

Data availability

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Disclosure statement

The authors declare no conflict of interest.

Additional information

Funding

This study is supported by National Natural Science Foundation of China (No. 81901582), Natural Science Foundation of Chongqing (No. cstc2021jcyj-msxmX0123) and Education Committee Science Foundation of Chongqing (No. KJQN201900417).