Introduction
Rheumatoid arthritis (RA) is a chronic autoimmune disease with obvious clinical symptom of persistent synovitis and joint damage [
1]. Dysregulation of multiple immune cells recognize autoantigens, generate autoantibodies and inflammatory cytokines which in turn promote autoimmune responses [
1,
2]. Activated immune cells infiltrate in joint tissue, facilitate the proliferation of synovial fibroblast and secretion of metalloprotease, finally result in progressive destruction of articular cartilage and subchondral bone [
2]. During the early stage of RA onset, the activated autoimmune response starts with the T-cell recognition of self-antigens expressed on antigen-presenting cells, and subsequently stimulates CD4
+ T cells to differentiate into T helper (Th) cells, arising unusually high Th17 population [
2]. The imbalance of regulatory T (Treg)/Th17 cell ratio then promotes the interaction of immune cells with synovial fibroblasts, and eventually aggravates joint inflammation [
2]. Moreover, Th17 cell also plays a crucial role in the pathogenesis of RA by producing IL-17 and other pro-inflammatory cytokines [
3]. Along with RA progression, it finally causes irreversible joint disability and other organ damage, such as heart, lungs, kidney, and even nervous and vascular systems [
1,
4]. Furthermore, the long-term use of disease-modifying anti-rheumatic drugs (DMARDs) such as Methotrexate (MTX), Leflunomide (LEF) and Cyclosporine A (CSA) for RA promotes the development of drug resistance, thus giving rise to refractory RA [
5]. The occurrence of refractory RA finally affects the quality of life in RA patients [
6], and therefore, it is of utmost importance to develop new treatment strategies based on understanding of RA pathogenesis.
p53 is known as a key tumor suppressor gene that regulates cell cycle arrest, DNA repair, apoptosis, etc. [
7]. p53 also plays a critical role in inflammatory and immune responses, responsible for expressions of multiple cytokines and matrix metalloproteinases (MMPs) [
8,
9]. Besides, p53 has been reported to mediate immune-related genes, such as CC-chemokine ligand 2 (CCL2), interferon regulatory factor 5 (IRF5), IRF9 and Toll-like receptor 3 (TLR3) [
10]. Given the importance of p53 in homeostatic regulation of immune responses, malfunction of p53, due to its deficient expression and mutation, exerts detrimental effect in the pathogenesis of cancer and autoimmune diseases [
11,
12]. Notably, clinical studies revealed that variable p53 mutation rate was found in cancer cells as well as synovial fibroblast [
13,
14]. These p53 mutations usually occurred at residues 94–312 located within central DNA binding domain (DBD), which reduced the binding affinity towards target gene promoters [
15,
16]. As a consequence, mutant p53 inactivates tumor suppressor function, promotes proliferation, invasiveness, immune evasion and development of multidrug resistance (MDR) [
11,
17,
18]. MDR1 was the first ATP-binding cassette (ABC) transporter associated with multidrug-resistance (MDR), which can be specifically activated by mutant p53 via ETS-binding site in MDR1 promotor region, thereby initiating the transcription of MDR1. Accordingly, drug resistance induced by mutant p53 mainly through ABC transporter-mediated efflux of drugs. Moreover, mutations of p53 have been reported to prevent drug-induced cell apoptosis, promote cells proliferation, activate DNA repair and inhibit autophagy to enhance microenvironmental resistance [
19,
20]. Apparently, these drug resistance and apoptosis-resistance mediated by mutant p53 might eventually drive the disease progression to drug resistant and refractory stage [
21,
22]. Although intensive studies have been carried out in cancer, the mechanism underlying p53 mutant-mediated MDR in RA is yet to elucidate.
Among various p53 mutations, R213* was identified as the most common of p53 nonsense mutation hotspot in cancer. For example, p53
R213* mutant upregulated IL-6 expression and downregulated Bax promoter activity in HS68 cells [
11,
20,
23]. p53
R213* also resulted in cell cycle arrest via downregulation of p21 expression in H1299 [
24]. Moreover, p53
R213* promoted the tumor growth and metastasis in Melanomas [
25]. In contrast to the aforementioned studies in cancers, the role of p53
R213* in the pathogenesis of RA remains unexplored. In accordance with our preliminary in vitro results, the most promising p53
R211* (human p53
R213*) mutant was selected for further examining its effect in adjuvant-induced arthritis (AIA) rats. Surprisingly, we unraveled that this mutant showed immunomodulatory effect in AIA rats, and the underlying mechanism of TBK1 inhibition was reported.
Materials and methods
Cell transfection with p53 wide type and mutants
Plasmids of human p53WT (GeneBank: NM_000546.6) and various mutations were constructed in our laboratory. RAFLS were transfected with p53WT and p53 mutants by using Lipofectamine® 3000 (Invitrogen). 5 × 105 RAFLS cell were seeded per well in a 6-well plate for overnight culture. 125 µl of Opti-MEMTM I medium, 4 µl of Lipofectamine 3000 reagent, 4 µl of P3000TM reagent and 2 μg DNA plasmid were added to each well, then changed the medium after 8 h. Cells were harvested after transfection for 48 h.
Cytotoxicity assays
All tested compounds were dissolved in DMSO to a final concentration of 100 mmol/L (HCQ, CSA), 500 mmol/L (FK506) and 1000 mmol/L (LEF) respectively. 6 × 104 RAFLSs were seeded per well in a 96-well plate. After overnight culture, the cells were treated with compounds for 72 h. 10 µl of MTT was added to each well. After incubation at 37 ℃ for 4 h, 100 µl of solubilization buffer (10% SDS in 0.01 mol/L HCI) was added and incubated for overnight. The OD values were measured at 575 nm wavelength (A575). The percentage of cell viability was calculated using the following formula: cell survival (%) = Atreated/Acontrol × 100. Data were obtained from three independent experiments.
RNA extraction and cDNA reverse transcription
Total RNA of frozen rat joint tissues was extracted using TRIzol (Invitrogen, USA). After quickly frozen in liquid nitrogen, approximately 40–60 mg crushed tissues were added in 1 ml of TRIzol. 200 µl chloroform was added and shaked vigorously, then the mixture was centrifuged at 12,000 rpm for 10 min at 4 °C. Supernatant and isopropanol was added in a ratio of 1:1.2 (supernatant:isopropanol) to a new tube, then centrifuged at 12,000 rpm for 10 min at 4 °C. After removal of supernatant, the precipitate was washed with 75% cold ethanol, and finally 50 µl of RNase-free water was added to dissolve the precipitate. UV spectrophotometry (NanoDrop Technologies, USA) was used to measure the quality and concentration of RNA. 1 μg of total RNA was reverse transcribed to corresponding cDNA using Maxima™ H Minus cDNA Synthesis Master Mix (Thermo, USA).
Real-time quantitative PCR
A total of 10 µl of PCR mixture consisted of 1 µl of cDNA, 0.2 µl of forward and reverse primers respectively, 0.2 µl of Dye, 5 µl of SYBR Master Mix (Roche Diagnostics, USA), and 3.4 µl of ddH2O. Quantification of gene expression was determined by ViiA 7 Real-Time PCR System (Applied Biosystems). Data were normalized to β-actin and analysed using the 2−ΔΔCT method.
Western blot
The cells were lysed with RIPA lysis buffer (1 × Protease inhibitor cocktail from Roche; 100 mM of PMSF, 150 mM of NaCl, 100 mM of DTT; 50 mM of Tris–HCl, pH 7.5; 0.5 Mm of EDTA) for 20 min. The soluble fractions were collected after centrifugation at 12,000 rpm for 15 min at 4 °C. Protein concentrations were measured using Bio-Rad protein assay. 30 µg of protein was separated by 10% SDS-PAGE then transferred onto PVDF membranes (Bio-Rad, USA) and blocked with 5% non-fat-dry milk for 1 h. These membranes were stained with primary antibodies overnight at 4 °C. After washing the membranes using TBST thrice, the membranes were incubated with anti-rabbit or anti-mouse secondary antibodies (1:2000; Santa Cruz Biotechnology) for 2 h at room temperature. ELC assay by FluorChem R (ProteinSimple, America) was used to visualize the hybridized bands under the Amersham Imager 600 (GE) Imaging System. Band densities quantification was analyzed by Image J software.
Establishment of adjuvant-induced arthritis (AIA) in SD rat and treatment
Male Sprague–Dawley (SD) rats weighing 80–100 g were purchased from SPF Biotechnology Co., Ltd (Beijing, China). Rats were divided into seven groups (n = 8–10) as following: (1) Healthy control (n = 8), (2) AIA + AAV-EGFP (Vehicle control, n = 8), (3) AIA + Methotrexate (MTX 7.6 mg/kg) (n = 8), (4) AIA + AAV-p53WT (1 × 1011 PFU for each joint) (n = 10), (5) AIA + AAV-p53WT + MTX (n = 8), (6) AIA + AAV-p53R211* (n = 10), (7) AIA + AAV-p53R211* + MTX (n = 8). pAAV-CMV-p53-3 × FLAG-EF1-mNeonGreen-WPRE (Ad-p53WT), pAAV-CMV-p53R211*-3 × FLAG-EF1-mNeonGreen-WPRE and vehicle control Adeno-associated virus (AAV) were constructed and packaged by OBiO technology (ShangHai, China). One week before AIA induction, 30 µl AAV-EGFP or AAV-P53R211* were injected into each knee-joint of each rat. For establishment of AIA model, mycobacterium tuberculosis (M. tuberculosis DES. H37 RA, DIFCO, USA) was emulsified in mineral oil (Sigma) to yield 2.5 mg/ml of M. tuberculosis. Rats were injected with 0.1 ml of this emulsion subcutaneously at the base of the tail on Day 0. Rats received MTX treatment (7.6 mg/kg) by gavage once a week. Arthritic scores and hind paw volume were evaluated and recorded every 3 days. On Day 28, these rats were sacrificed, blood, organs, and joint tissue were harvested for biochemical assays and micro-CT analysis.
Assessment of joint swelling and arthritic scores
The volume of hind paw and arthritic scores were evaluated every 3 days. The investigator was blinded to the group allocation when measuring the joint swelling. The volume of hind paws was measured using Plethysmometer (Ugo Basile, Italy or Kent Scientific Corp, Connecticut USA). Clinical scores were obtained for each paw according to the following standards the severity of arthritis was objectively inspected on four paws and was scored on a scale of 0–4 for each paw according to the arthritis index: score 0 means there was no swelling and erythema evidence on the joint, including the small joints of the front foot and phalangeal joint, large joints including wrist and ankle; score 1 means there was mild swelling and erythema on ankle; score 2 means mild swelling and erythema extended to small joint; score 3 means there was serious swelling and erythema on large joint; score 4 means serious swelling and erythema encompassing on small and large joint.
Micro-CT analysis
At the end of treatment period, the right hind paw of sacrificed rats was amputated and fixed in 4% PFA, then scanned using in vivo micro-CT scanner (SkyScan 1176, Bruker, Belgium). The following scanning parameters were used to obtain high-quality images of the rat joint: 35 µm resolution, 85 kV, 385 µA, 65 ms exposure time, 0.7, rotation step in 360°, and a 1 mm Al filter. The images were reconstructed using NRecon software (Bruker-micro-CT, Belgium). Bone density was analyzed by CT An software. Micro-CT score was calculated from five disease-related indexes of the micro-CT analysis for calcaneus, including bone mineral density, bone volume fraction, cortical mineral density, trabecular number, and total porosity using the following formula: (Acquired value − minimum value)/(maximum value − minimum value) or 1-(Acquired value − minimum value)/(maximum value − minimum value). The final micro-CT score is equally averaged from the above five aspects of bones (Micro-CT score: 0–0.2, mutilating; 0.4–0.6, moderate; 0.8–1, normal). According to the severity of bone erosion, the bone condition of rats was evaluated with radiological score.
Hematoxylin and eosin (H&E) and immunofluorescent (IF) staining
Joint tissues were fixed in 4% paraformaldehyde for 24 h and subjected to dehydration. The tissues were embedded in paraffin for microtome sectioning and H&E or IF. Joint sections (6 μm thickness) were dehydrated, deparaffinized and rehydrated. For H&E staining, joint tissues were subjected to treatment with hematoxylin and eosin. For IF, joint tissues were incubated with primary antibody at 4 ℃ overnight. The secondary antibody (anti-rabbit FITC, anti-mouse Cy5, CST) was incubated at room temperature in the absence of light for 2 h. The coverslips were mounted with FluorSave™ Reagent (Millipore, USA). H&E images were captured by light microscopy (Leica DM2500, Germany). Fluorescent images were captured by API Delta Vision Live-Cell Imaging System.
Flow cytometric analysis of lymphocytes
Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of rats using a standard Ficoll density-gradient centrifugation kit (GE Healthcare). The collected spleen was washed with PBS and grinded to collect lymphocytes. T cells were stained with the following antibodies: anti-rat CD45 (APC/Cyanine7); anti-rat CD3 (FITC); anti-rat CD4 (PerCP/Cyanine5.5); anti-rat CD8a (APC). For Treg cell analysis, cells were stained with anti-CD3, anti-CD4, anti-CD8, and anti-CD45 antibodies, then were washed, fixed, and permeabilized prior to staining with anti- Foxp3 (PE) antibody. For Th17 cell analysis, after stained with surface markers (anti-CD3, anti-CD4, anti-CD8, and anti-CD45), cells were fixed and permeabilized prior to staining withanti-IL-17A antibody (PE-Cyanine 7). All samples were measured by FACSAria III flow cytometer (BD Bioscience) and analyzed using the FlowJo v10 software (TreeStar, Inc.).
LEGENDplex assay kit
Serum was collected from rat blood by centrifuging it at 3500 rpm for 10 min. The concentration of IL-10, IFN-γ, KC, TNF-α, IL-18, IL-12p70, IL-1β, IL-17A, IL-1α, GM-CSF, IL-33 and IL-6 in rat serum were determined using LEGENDplex (Biolegend, USA) kit according to the manufacturer’s instruction. The samples were analyzed by FACSAria III flow cytometer (BD Bioscience) and LEGENDplex ™ Data Analysis Version 8.0 software.
Immunoprecipitation
The antibody (5 µg) was vortex-mixed with 50 µl immune-magnetic bead (Thermo, USA) and incubated at 4 ℃ overnight. The extracted protein was then added to the mixture and incubated at 4 ℃ for another overnight. Subsequently, the bead mixture was transferred to a clean tube and placed in a magnetic separation rack. The supernatant was discarded, and the residue was washed with washing buffer by using magnetic separation. The Dynabeads-Ab-Ag complex was re-suspended in 60 µl of elution buffer. 20 µl of 5 × loading buffer was added, and the resulting mixture was incubated for 10 min at 100 °C. The supernatant was separated by magnetic separation and stored at − 80 ℃.
ELISA
Cytokines expressions in rat serum (50 µl for each sample) were detected by using specific Quantikine enzyme-linked immunosorbent assay (ELISA) kit (MEIMIAN, China) according to manufacturer’s instruction. The absorbance was measured at 450 nm using SpectraMax Paradigm (Molecular Devices, USA).
Nuclear and cytoplasmic extraction
The transfected cells were harvested and transferred to a clean tube, followed by centrifugation at 500g for 5 min. Ice-cold CER I was added to the cell pellet and incubated on ice for 10 min. The cell extract was then vortex-mixed for 15 s, followed by centrifugation at maximum speed for 5 min. The supernatant (cytoplasmic protein) was transferred to a clean ice-cold tube. After that, ice-cold NER was mixed with insoluble residue in the tube, and the tube was placed on ice with vortex-mixing for 15 s at an interval of 10 min for 40 min in total. Subsequently, the tube was centrifuged at maximum speed for 10 min and transferred the supernatant to a new ice-cold tube (nuclear protein). Both extracts were stored at − 80 ℃.
Total RNA sequencing and analysis
Collected rat knee joint were sent to LC-BIO Technologies Co., LTD (Hangzhou, China) for RNA extraction, library preparation and sequencing on Illumina NovaSeq 6000 plateform, following their standard procedure. DEGs analyses were performed by DESeq2 1.30.1 [
26], after interpretation of DESeq2, DEGs were screened from the data with
p value < = 0.05 and fold change > = 2 or foldchange < = -2. The distribution of data was presented in a volcano plot which builds by the plot function in R 4.2.1 [
27]. KEGG enrichment analysis was performed by R package ClusterProfiler v3.18.1 [
28]. The p-value Cut-off for enrichGO was set to 0.05. DEGs data were further input into the KEGG gene tools [
29] and converted into KEGG Orthology ID [
30], the converted IDs combined with corresponding gene symbols were entered into the KEGG Mapper–Reconstruct tools [
31] to identify the respective classifications, to visualize the classification analysis results, ggplot2 package (version 3.3.6) [
32] were applied in R 4.2.1 [
27] for the construction of KEGG Classification chart. STRING analysis was performed using Multiple Protein modules. After the network generated by STRING, while all other parameters were statically unchanged, the active interaction sources were set as only the Experiments and Database (which was denoted as Curated Database at the retrieve version of STRING).
Statistical analysis
T test was used to analyze two groups. Over 3 or more groups were analyzed by one-way analysis of variance. All results are expressed as the means ± SEMs. GraphPad Prism 7.0 software was used to evaluate the statistical data. p < 0.05 was considered significant.
Role of funding source
Funders of this study did not have any role in study design, data collection, data analysis, data interpretation, or writing of the report.
Discussion
In this study, we observed the experimental differences, in which p53 mutant exhibits immunomodulatory effect without resistance to MTX in vivo (Figs.
2 and
3) but not observed in vitro (Supplementary Table
S1). Rheumatoid arthritis fibroblast-like synoviocyte (RAFLS) derived from synovial tissue of RA patients has been widely used as a representative cell line for studying inflammation, apoptosis and cell proliferation in RA research [
54]. Although RA is symptomized by chronic inflammation in joint, its pathogenesis originates from systemic immune abnormality [
55]. Therefore, using a single in vitro cell model alone is inadequate to simulate the complicated in vivo immune environment of RA [
56]. Discrepancies are commonly observed between in vitro and in vivo studies, especially in immunological studies [
57]. For instance, bone marrow-derived mesenchymal stem cells (MSC) suppressed T cell proliferation in vitro, while MSC treatment did not show immunomodulatory effect via inhibiting T cell proliferation nor ameliorate the disease severity in collagen-induced arthritis (CIA) mice [
58]. Another study demonstrated that CD3-peptides had no effect on T cell function in vitro but unexpectedly repressed inflammatory responses in AIA rats [
56]. Moreover, adiponectin (AD) had an obvious anti-inflammatory function in CIA mice [
59], but previous data suggested that AD increased IL-6 expression in synovial fibroblasts [
60]. Such experimental discrepancies were also observed in other diseases related immune dysfunction such as diabetes mellitus. Diabetic db+/db+ mice showed defects in immune functions in terms of delayed skin graft rejection and a significant increase in plaque-forming cell (PFC) response to sheep erythrocytes (SRBC) [
61]. However, contrary in vitro results were obtained with spleen cells isolated from those db+/db+ mice, where negligible fluctuations were observed in responses to allogeneic cells and SRBC-induced PFC. Other than the intricate immune responses, stiffness of cell environment affects biomechanical and physiochemical properties of cells, becoming another factor for in vitro and in vivo discrepancies [
62]. Together with these cited examples, our study well explain the importance of in vivo study that provides a more comprehensive view for examining disease pathogenesis, although in vitro studies are still irreplaceable due to low cost and high efficiency.
Apart from p53
R213* mutant, p53
WT also exhibited a similar anti-inflammatory effect in AIA rats (Fig.
2A–C); however, p53
WT showed no interaction with TBK1 (Fig.
6C), implying a different anti-inflammatory mechanism. In fact, p53
WT has been extensively reported to regulate the expressions of inflammatory cytokines and Matrix metalloproteinases (MMPs) via various pathways [
63]. Overexpressed p53
WT suppressed the expression of IL-6 in AIA rats, via inactivation of nuclear factor kappa-B (NF-κB), Mitogen-activated protein kinase (MAPK) and Extracellular regulated protein kinases (ERK) pathways [
64]. Specific dephosphorylation of p38 MARK by WIP-1 phosphatase was mediated by p53
WT [
65]. Moreover, overexpression of p21, the downstream gene of p53
WT, also declined the expressions of IL-6 and MMP-1 in RAFLS, via Jun kinase/Activator protein-1 (JNK/AP-1) pathway [
66]. Apparently, in vivo study further proved that p53-deficient mice model exacerbated the severity of arthritis with increasing expressions of collagenase-3 and pro-inflammation cytokines [
67]. Meanwhile, p53 function is highly associated with the homeostasis of innate and adaptive immune responses. Deficiency of p53 in RA downregulated Toll-like receptors expressions and modulates T cell differentiation, and ultimately triggered chronic inflammation [
63]. This proposes that p53
R213* mutant exhibits anti-inflammation in RA, independent of p53
WT-mediated pathway.
As a central mediator of innate immune defense system, TBK1 activates downstream genes for production of type I interferons (IFNs) and other inflammatory cytokines against pathogenic infection [
68]. However, overactivation of TBK1 is closely related to dysregulation of immune responses, which is key pathogenesis of autoimmune diseases [
69]. For example, elevated TBK1 expression was commonly observed in autoimmune diseases including systemic lupus erythematosus (SLE) [
70], primary Sjögren’s syndrome (pSS) and systemic sclerosis (SSc) patients [
71]. Patients with RA also showed upregulation of TBK1 downstream IFN-I expression [
72]. Similarly, our results also revealed a high expression level of pTBK1 (Fig.
7A), overactivation of CD4
+ cells (Fig.
3A) and infiltration of Th17 cells (Fig.
7B) in AIA rats. Likewise, TBK1-deficient FLS revealed the suppression of IFN-β and IP-10 expressions [
73]. All these results suggest the pathogenic role of TBK1 in autoimmune diseases including RA, thus proposing TBK1 as a potential therapeutic target. Indeed, the use of pyrimidine-based TBK1 inhibitor was found to downregulate IFN expression in vitro and in vivo [
74]. Another study also reported that TBK1 inhibitor greatly delayed the onset and decreased the severity of experimental autoimmune encephalomyelitis (EAE), and even suppressed the relapse of EAE [
75]. Moreover, treatment with TBK1/IKKε inhibitor reduced IFN-I level in PBMCs extracted from pSS, SLE and SSc patients [
71]. Herein, we also reported that binding of TBK1 with p53
R213* mutant inhibited TBK1-STING-IRF3 cascade, thereby exhibiting immunosuppressive and anti-inflammatory effects in AIA rats. These results well demonstrate TBK1 as a promising drug target for RA, although no TBK1 inhibitor has been approved for RA treatment [
76]. Therefore, the development of small molecules or small peptides as TBK1 inhibitor provides a prospective strategy for the treatment of RA or even other autoimmune diseases.
P53 mutants, such as R248W, R249S, R273H and R280K, have been reported to intervene cyclic GMP-AMP synthase (cGAS)-STING-TBK1-IRF3 immune cascade, resulting in immune evasion and thus promoting tumorigenesis in cancer[
18]. Similarly, we also clarified that human p53
R213* (rat p53
R211*) suppressed immune response in RA mainly via blocking TBK1-IRF3-STING innate immune pathway. Although variable outcomes of p53-mediated immune response in cancer and RA development were observed, we propose that the effect of p53 mutants on immunity may share similar mechanism. For instance, mutant p53 correlated with upregulation of programmed cell death-Ligand 1 (PD-L1) which facilitated cancer cells to escape from immune system [
77,
78]. However, in autoimmune disease, PD-L1 showed an opposite outcome by suppressing autoreactive T cell function [
79]. Another representative example is that mutant p53 compromises the transcription of immune-related Toll-like-receptor 3 (TLR3) in cancer cells [
80,
81]. And conversely, inhibition of TLR3 attenuates the symptom of RA [
82]. Of note, TLR3 activation is responsible for inflammatory status, promotion of osteoclast differentiation and expressions of B cell survival/proliferating factors B cell activating factor (BAFF) and aspartate aminotransferase-to-Platelet Ratio Index (APRI) in RA [
83‐
85]. Moreover, mutant p53 induces suppression of macrophage activation and promotes immunosuppressive Treg cell via expression of Transforming growth factor beta (TGF-β), thereby facilitating tumor progression [
86]. Conversely, in RA condition, macrophages are usually over-activated, and the differentiation and function of Treg cells are commonly suppressed [
87]. One more example is that antigen presentation triggers abnormal differentiation of autoreactive T cells and accelerates autoimmune response in RA [
88,
89]. Alternatively, major histocompatibility complex class I (MHC-I) related antigen presentation was retarded by mutant p53 in cancer [
78]. In brief, mutations of p53 promote cancer pathogenesis by mediating immune evasion and proliferation of tumor cells, while these immune escape mechanisms may impose opposite consequences in RA. We here demonstrated that human p53
R213* (rat p53
R211*) attenuated the disease severity mainly by its immunomodulatory effect in RA. In addition to the blockage of TBK1-IRF3-STING pathway, mutant p53
R211* may exhibit immunomodulatory effect through aforementioned mechanism. Given that the effect of different p53 mutations on RA pathogenesis has not been well explored so far, further studies are required to unravel the detailed mechanism of action.
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