Introduction

Rheumatoid arthritis (RA) is a chronic inflammatory polyarthritis characterized by marked inflammation and hyperplasia of the synovial membrane, which might also involve extra-articular tissue. The detailed etiology is still obscure, but genes and environmental factors, including bacterial and viral infection, could be involved in developing RA1although the pathogenic mechanisms that link virus infection and RA are still unknown. Parvovirus B19 and retroviruses could infect the synovial membrane directly, while Epstein-Barr virus might induce RA due to molecular mimicry2.

Osteoarthritis (OA) is a chronic degenerative arthritis related to aging, biomechanical loading, obesity, and genetics, where the hallmark of OA is pathologic changes to articular cartilage, and although variable synovial inflammation occurs in most OA patients, the extent and severity of synovial inflammation appear less than in RA; clinically, the major differences between RA and OA are the severity of synovial inflammation, affected joints, etiologies, and treatment regimens3.

Parvovirus B19 is a small, single-stranded DNA virus. The clinical manifestations of B19 infection include cutaneous, hematological, articular, neurological, and cardiovascular disease4,5,6,7,8. Parvovirus B19 infection appears to induce acute symmetric polyarthritis resembling RA, and this acute arthritis can progress to be chronic and erosive, fulfilling the classification criteria of RA9. Besides RA, several other autoimmune diseases might be related to parvovirus B19 infection, such as juvenile idiopathic arthritis, systemic lupus erythematosus, systemic sclerosis, psoriasis, anti-phospholipid syndrome, and vasculitis10,11.

Takahashi et al. demonstrated that parvovirus in RA synovial tissue was infective, and could upregulate IL-6 and TNF-alpha expression12. They concluded that B19 was involved in the initiation and perpetuation of synovitis in RA patients, and was a causative agent in the development of RA. Ray et al. showed induction of an invasive phenotype of normal synovial fibroblasts by parvovirus B1913. Our previous study demonstrated that anti-parvovirus B19-IgG, -IgM antibodies, and B19 DNA could be detected in RA patients’ plasma and synovial fluid14. The non-structural protein 1 (NS1) and viral capsid protein 1 (VP1) of B19 could also be detected in the synovial membrane of RA patients.

Accordingly, parvovirus B19 could play a role in the pathogenesis of RA, but as this role in the development of inflammatory arthritis is still controversial15, this study aimed to investigate whether parvovirus B19 could induce inflammation in synovial membranes mimicking RA.

Results

The top five molecular pathways involved after transduction of LEX-NS1 into the SW982 cell line

The Ingenuity Pathway Analysis (IPA) calculates the z-score16. A positive z-score indicates increased pathway activity, while a negative z-score indicates decreased pathway activity. The acute phase response, nuclear factor erythroid 2-related factor 2 (NRF2)-mediated oxidative stress response, and mTOR signaling pathways were activated with a z-score ≧ of 2, whereas interferon (IFN) and factor-promoting cardiogenesis in vertebrate signaling pathways were suppressed, with a z-score ≦ of – 2, after transduction of LEX-NS1 into the SW982 cell line (Fig. 1). The activation or inhibition in all these pathways is significant with a -log (p-value) > 1.31.

Fig. 1
figure 1

The top 5 molecular pathways involved after transduction of LEX-NS1 into the SW982 cell line. Brown bar: activation; Blue bar: inhibition; vertical line: -log (p-value) = 1.31. These pathways have an absolute z-score ≧ of 2.

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Activation of the acute phase response signaling pathway

The acute phase response signaling pathway was activated (Fig. 2). In this pathway, many inflammation-related or pro-inflammatory cytokines promoting molecules including IL-6, TNFRSF1A associated via death domain (TRADD), evolutionary conserved signaling intermediate in Toll pathways (ECSIT), nuclear factor-IL6 (NF-IL6) also known as CCAAT enhancer binding protein beta (CEBPB), serum amyloid A (SAA), C3, heme oxygenase 1 (HMOX1 or HO-1), ferritin and haptoglobin (HP) were significantly upregulated with the consequent up-regulation of these molecules inducing inflammation of the synovial membrane.

Fig. 2
figure 2

The cascaded molecules are involved in the acute-phase response signaling pathway. Red: up-regulated molecules in this study; Green: down-regulated molecules.

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Inhibition of the IFN signaling pathway

IFNs are cytokines that play roles in infection, inflammation, and autoimmunity. The IFN signaling pathway was inhibited (Fig. 3). In this pathway, the signal transducer and activator of transcription 1 (STAT1), IFN-induced transmembrane protein 1 (IFITM1), IFN-induced protein with tetratricopeptide receptor 3 (IFIT3), and MX dynamin-like GTPase 1(MX1) were downregulated, although suppressor of cytokine signaling 1 (SOCS1) was upregulated.

Fig. 3
figure 3

The cascaded molecules are involved in the interferon signaling pathway.

Red: up-regulated molecules in this study; Green: down-regulated molecules.

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Activation of the NRF2-mediated oxidative stress response pathway

Oxidative stress plays a role in joint destruction in RA patients. The activation of the NRF2-mediated oxidative stress response signaling pathway was demonstrated in this study (Fig. 4). The ubiquitin-conjugating enzyme E2 R1 (UBE2R1) also known as cell division cycle 34 ubiquitin-conjugating enzyme (CDC 34), HO-1, ferritin light chain (FTL), ferritin heavy chain 1 (FTH1), epoxide hydrolase 1 (EPHX1), peptidyl proline isomerase B (PPIB), Ras, MAPK/ERK kinase 2 (MEK2) also known as mitogen-activated protein kinase kinase 2 (MAP2K2), and JunD proto-oncogene, an AP-1 transcription factor subunit, were all significantly upregulated, thereby assisting in maintaining oxidant/anti-oxidant homeostasis.

Fig. 4
figure 4

The cascaded molecules are involved in the NRF2-mediated oxidative stress response pathway. Red: up-regulated molecules in this study; Green: down-regulated molecules.

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Activation of the mTOR signaling pathway

The mammalian target of rapamycin (mTOR) signaling pathway plays a role in regulating cell metabolism, growth, proliferation, and survival, and is activated during angiogenesis and T-cell activation, which are common findings in the synovial membrane of RA patients. Activation of the mTOR pathway was shown in this study (Fig. 5). The eukaryotic 4E binding protein 1 (4EBP1), also known as eukaryotic translation initiation factor 4E binding protein 1 (EIF4EBP1), ras homolog family member T2 (RHOT2), and RAS were significantly upregulated.

Fig. 5
figure 5

The cascaded molecules are involved in the mTOR signaling pathway. Red: up-regulated molecules in this study; Green: down-regulated molecules.

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Gene expressions in this study were like those in the RA dataset rather than the OA dataset

Hierarchical clustering was used to compare the z-scores of pathway activity among this study (NS1_RNA-seq), two RA datasets (GSE55235, GSE77298), and two OA datasets (GSE55457, GSE206848) (Fig. 6), which showed similarities among the datasets. In all pathways, the pathway status in this study is closer to the RA dataset (GSE77298) than to the OA datasets (GSE206848, GSE55457). Moreover, similar findings could also be found in the cellular immune, humoral immune, and growth factor pathways. In the cytokine, apoptosis, cellular growth, and cellular stress pathways, the pathway statuses in this study are closer to the RA datasets (GSE77298, GSE55235) than to the OA datasets (GSE206848, GSE55457).

Fig. 6
figure 6

Comparisons of activation or inhibition in various pathways among B19-NS1 transduced into the SW982 cell line and the GEO datasets (RA and OA). Red: activation; Blue: inhibition. Hierarchical clustering was used to compare the z-score of pathway activity among this study (NS1_RNA-seq), 2 RA datasets (GSE 55235, GSE 77298), and 2 OA datasets (GSE 55457, GSE 206848). For a clear view, the photos of these pathways were cropped. The full-sized photos have been attached.

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The pathway activities, including all immune-related pathways, in this study were closer to RA than those in OA (Table 1). These results revealed that the gene expressions after transduction of parvovirus B19-NS1 into a synovial cell line were similar to those in RA patients.

Table 1 Datasets with similar pathways.
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Discussion

This study demonstrated the activation of the acute phase response, oxidative stress response, and mTOR signaling pathways, as well as the inhibition of the IFN signaling pathway after transduction of B19-NS1 into the SW982 cell line.

The synovial membrane contains fibroblast-like and macrophage-like cells. In this study, we wished to investigate whether the B19-NS1 protein induces immune and inflammatory responses like RA. A cell line derived from RA, already possessing the characteristics of RA, is not suitable for this study. Therefore, we used an SW982 cell line, which has a mixed type of cells and was derived from a human synovial sarcoma patient, not from an RA patient17,18.

Several inflammation-related molecules such as IL-6, TRADD, SAA, Ho-1, and HP were upregulated in the acute phase response pathway. The upregulation of IL-6 by B19-NS1 has been validated in several previous studies19,20 where NS1 acts as a transcriptional factor of IL-6 gene, and induces the expression of IL-620, being an important cytokine in RA, and the treatments targeting IL-6 have a good response in clinical practice21,22. TRADD is highly expressed in RA patients23 and is in the cascade of the TNFR1 signaling pathway, which activates several inflammatory pathways, including NF-kB, p38 MAP kinase, and apoptotic signaling24, so treatment targeting TRADD might hinder the progression of RA25. TNF blockers are widely used in the treatment of RA nowadays26,27. SAA is an acute-phase protein synthesized in adipose tissue and the liver, and is elevated in active RA patients, correlating with disease activity28,29. NF-IL6 is a nuclear transcription factor that regulates the expression of various genes involved in cell differentiation, proliferation, and immune function30,31. It can be detected in the synovial lining, macrophage-like, and fibroblast-like cells in RA patients, and is involved in cartilage degradation while being correlated with the chronic inflammation in RA patients32. NF-kB is activated in macrophage-like and fibroblast-like cells, promoting joint destruction33. ECSIT regulates innate immune response by activating the NF-kB signaling pathway and promotes pro-inflammatory cytokine production34; furthermore, it also regulates the synthesis of mitochondrial reactive oxygen species during infection, which is harmful to the tissues35.

The NRF2-mediated oxidative stress response pathway was activated in this study. HO-1 is expressed in response to pro-inflammatory cytokines and oxidative stress; and plays a protective role against inflammation36, and while being dependent on the activity of transcriptional activator NRF2. Its upregulation in the synovial fluid of RA patients exerts an anti-inflammatory effect and maintains oxidant/ antioxidant homeostasis37,38. UBE2R1 is a ubiquitin-conjugating enzyme involved in the ubiquitin-proteasomal pathway. The dysregulation of the ubiquitin-proteasomal pathway caused by oxidative stress results in pro-inflammatory cytokines production and exacerbates RA39. Arthritis patients have oxidative stress in their joints, producing epoxide in the synovial membrane. EPHX1 is expressed on the macrophage-like and fibroblast-like synoviocytes, which catalyzes the hydroxylation of epoxides to less reactive, more water-soluble, and easily excretable trans-dihydrodiols40, and while playing a role in the detoxification of reactive epoxide. Its up-regulation prevents reactive epoxide-induced tissue damage in arthritis patients, which might be a self-protection mechanism41,42. PPIB, also known as cyclophilin B, is a cyclosporine-binding protein as a cell receptor, which might regulate cyclosporine-mediated immunosuppression43. It regulates protein conformation via isomerization in the endoplasmic reticulum (ER), participating in protein folding, secretion, and post-translational modification. Consequent ER stress caused by abnormal unfolded or misfolded protein accumulation can induce inflammation accordingly, with PPIB being involved in the inflammatory process44. Extracellular cyclophilin also plays a role in the pathogenesis of inflammatory diseases, as the interaction between extracellular cyclophilin and its receptor, CD147, results in chemotaxis and production of pro-inflammatory cytokines45. Ferritin, composed of FTL and FTH, is a Fe+ 2 storage protein in ferroptosis, which is related to the pathogenesis of RA, with both FTL and FTH able to be detected in fibroblast-like and macrophage-like synoviocytes46,47. This study demonstrated an increased FTH1 expression in the synovial membrane of RA patients, which was compatible with Ling’s report48.

4EBP1 is a member of the mTOR signaling pathway that is activated in rheumatoid synovitis49 and regulates the invasive properties of fibroblast-like synoviocytes in RA patients50, with its upregulation suggesting that the B19-NS1 may induce synovitis like RA, as the mTOR pathway is a potential target for RA treatment51.

Inhibition of the IFN signaling pathway was noted in this study. Upregulation of SOCS1 was noted. Isomaki et al. showed that SOCS1 was upregulated in the synovial membrane of RA patients52. Our previous study also demonstrated increased expression of SOCS1 in the peripheral blood mononuclear cells of RA patients53. STAT1 was downregulated in this study. The expression of STAT1 in RA patients varies after stimulation with different types of IFN. Kasperkvitz et al. showed increased IFN-β-induced STAT1 protein expression in RA synovium54; however, in RA patients, Sharma et al. demonstrated diminished STAT1 and IFN-γ-induced STAT1 phosphorylation in CD4 + T-cells55. Decreased phosphorylation of IFN-α-induced STAT1 was also shown by Ptacek et al.56. MX1 is induced by type I and II IFN, which antagonizes the replication process of several DNA and RNA viruses, and is related to cytokine signaling in the immune system57. MX1 expression was downregulated in this study. He et al. also showed downregulation of MX1 expression in RA synovial fibroblasts from a GEO dataset58.

In the comparison analyses, the pathway status after transduction of B19-NS1 into a synovial membrane cell line is like that found in RA synovium.

In conclusion, this study shows that B19-NS1 induces inflammatory responses in a synovial membrane cell line. Patients with parvovirus B19 infection might exhibit chronic progressive symmetric polyarthritis as RA. Our previous study showed that parvovirus B19 infection plays a role in the pathogenesis of RA in Taiwan. Taken together, parvovirus B19 infection could be one of the trigger factors in the development of RA.

Materials and methods

Cloning of pLEX-MCS-NS1 vector

The Parvovirus B19 NS1 DNA (GenBank: AB030673.1) was synthesized by Invitrogen GeneArt (Thermo Fisher Scientific). During the synthesis of B19-DNA, the myc was added to the 3’-end of NS1 as the tag. Then B19-NS1 DNA was cloned into LentiORF pLEX-MCS vector (Open Biosystem Inc.) and transformed into the stbl3 competent cells to obtain enough pLEX-MCS-NS1 vectors. The cloned vector pLEX-MCS-NS1 was also transfected into the 293FT cell, and a Western blot was performed to validate the expression of NS1-myc fusion protein with an anti-myc antibody. The pLEX-MCS-NS1 and Trans-Lentiviral pLEX packaging system (Open Biosystems Inc.), including packaging plasmid (psPAX2 gag-pol vector) and envelope plasmid (pMD2G packaging vector), were co-transfected into 293FT cells by using the calcium-phosphate precipitation method to produce LEX-NS1 lentiviral stock, with the pLEX-MCS vector itself being used as the control in this experiment. A similar packaging procedure was also performed to produce LEX lentivirus.

Transduction of LEX-NS1 lentivirus to the SW982 cell line

The LEX-NS1 lentivirus, containing a puromycin resistance gene, was transduced into three separate SW982 cell lines (a synovial sarcoma cell line, purchased from ATCC) in the presence of polybrene and selected with puromycin during culturing, while similar procedures were also performed with LEX lentivirus in three separate SW982 cell lines. Western blot confirmed the successful transductions, and the stable clones were prepared for further RNA-seq analysis.

RNA-seq

Total RNA was extracted from the transduced SW982 cell lines using Trizol reagent. SureSelect Stranded-Specific RNA library preparation kit (Agilent Technologies) was used for library construction and sequenced using Illumina’s sequencing-by-synthesis technology (Illumina, USA), while HISAT2 aligner was used for alignment. Differential expression analysis was performed using Cuffdiff, a tool for detecting differentially expressed genes in RNA-seq59. Genes with p-value < 0.05 and ≧ 2-fold changes were considered significantly differentially expressed.

Pathway analysis and comparisons of pathway activities with the GEO datasets

IPA was used for pathway analysis enriched by differentially expressed genes (DEGs) and comparisons of various pathway activities between this study and the GEO datasets.

Several datasets were downloaded from the GEO database. GSE77298 dataset contains the mRNA expression data of the synovial membrane in 16 RA patients and 7 healthy controls. GSE55235 and GSE55457 include mRNA expression data of the synovial membrane in 20 healthy controls, 26 osteoarthritis (OA) patients, and 33 RA patients. GSE206848 had mRNA expression data of the synovial membrane in 7 healthy controls, 7 OA patients, and 2 RA patients. DEGs in the synovial membrane were calculated in RA or OA patients compared to healthy controls in these datasets. The pathway activities enriched by these DEGs in these GEO datasets were then demonstrated in IPA and the pathway activities between LEX-NS1 transduced into the SW982 cell line and those in RA or OA patients’ synovial membrane were compared.

Z-score was calculated by the IPA software16and hierarchical clustering was used to compare the z-score of pathway activity in different datasets using IPA software.