About 30–50% of patients are unresponsive to conventional DMARDs. If a 2–6 month treatment with MTX mono- or combinational therapy is inadequate, biologic DMARDs should be added ^[36][43][36,43]. Biologic DMARDs include tumor necrosis factor (TNF) inhibitors, costimulation modifiers, IL-6 inhibitors and B cell depleting drugs. Commercially available biological drugs, such as etanercept (Enbrel^®), infliximab (Remicade^®), adalimumab (Humira^®), golimumab (Simponi^®) and certolizumabpegol (Cimzia^®) are all TNF inhibitors that block cytokine signaling, reduce cell recruitment, normalize IL-6 expression level in serum and matrix metalloproteinase (MMP) expression levels in cartilage and bone, and as a result, slow bone destruction. Among the aforementioned biologic DMARDs, TNF inhibitors should be the initial drugs used in cases with an inadequate response to conventional synthetic DMARDs, and are often used in combination with other DMARDs, especially MTX ^[44][45][44,45]. Nonetheless, these drugs have serious side effects, such as increased risk of infections and neurological diseases, development of multiple sclerosis and lymphoma ^[46][47][46,47]. Additionally, TNF inhibition has previously resulted in the development of skin tumor Merkel cell carcinomas in patients affected by rheumatologic diseases [48]. Clinical trials with TNF inhibitors have revealed that a number of patients did not respond to treatment [49]. In this case, another DMARD, anakinra, that binds to IL-1 receptors and blocks inflammation, is considered for therapy. Anakinra is used in combination with other DMARDs or as a monotherapy, but the application is limited due to the risk of opportunistic and latent infections ^[50][51][50,51]. Anti-CD20 monoclonal antibody, rituximab, depletes B cells and is a typical medication for the treatment of lymphomas, leukemia and autoimmune disorders, and in RA patients, it can be added when there is an insufficient response to TNF inhibitors [52]. T cell activation can be blocked by abatacept, which is the fusion protein containing the domain of cytotoxic T lymphocyte-associated antigen 4 and prevents T cell activation by binding to CD80 and CD86 receptors on antigen-presenting cells (APCs), as well as blocks interaction between DCs and T cells. A clinical study of abatacept demonstrated significant results, however some patients were insensitive to this treatment, which was associated with the loss of CD28 expression on T cells ^[53][54][53,54]. Another biologic DMARD, tocilizumab, blocks IL-6 receptors and significantly reduces disease severity in RA patients who have not been effectively treated with traditional DMARDs [55]. A clinical study of anti-IL-17 antibody, secukinumab, and anti-IL-17RA antibody, brodalumab, has shown low response in RA patients in both cases ^[56][57][56,57]. Therapeutic efficacy of biologic DMARDs, when used as a monotherapy, is less effective compared to the combination with MTX [58].

It was shown that cytokines, such as TNF-α, IL-1, IL-6, IL-7, IL-15, IL-17, IL-18, IL-21, IL-23, IL-32, IL-33 and granulocyte-macrophage colony-stimulating factor (GM-CSF) were implicated in the pathogenesis of RA [59]. However, clinical trials with therapeutic strategies blocking IL-1, IL-18 or IL-17 have shown few benefits. On the other hand, TNF-α or IL-6 targeting therapy was successful in relieving symptoms and initiating disease remission [60]. Another approach in RA therapy is targeting small molecules. The Janus kinases (JAK) inhibitors are a type of targeted synthetic DMARD that recognize and regulate the activity of the JAK family of non-receptor tyrosine kinases, which transduce signals from several different cytokine receptors through the effects on the STAT family of transcription factors. Tofacitinib represents a targeted synthetic DMARD that inhibits IL-6 production by blocking JAK1 and JAK3 through the IL-6/gp130/STAT3 signaling pathway [61], and as a result, inhibits IL-17 and interferon-γ (IFN-γ) production and the proliferation of CD4+ T cells in patients with RA ^[62][63][62,63]. Another targeted synthetic DMARD, baricitinib, a JAK1/JAK2 inhibitor, was superior compared with the TNF-α antagonist adalimumab in patients with an inadequate response to the MTX [64]. Upadacitinib, which is a JAK1 inhibitor, significantly improves the efficacy of RA treatment in patients who are unresponsive to MTX or a TNF-α antagonist [65]. Thus, targeted synthetic DMARDs should be considered as a monotherapy or in combination with conventional synthetic DMARDs [66].

NSAIDs alongside GCs are commonly used as adjuvants to basic therapy. They are applied to decrease pain and inflammation during RA, however NSAIDs are not able to reduce bone and cartilage destruction [67]. NSAIDs are typically divided into two groups based on their chemical structure and selectivity: a group of non-selective NSAIDs, which inhibit both cyclooxygenase-1 (COX-1) and COX-2 and another group of COX-2 selective inhibitors. COX-1 plays a role in maintaining gastrointestinal mucosa lining, kidney function and platelet aggregation, whereas COX-2 is expressed during an inflammatory response. The most common NSAIDs applied in RA include acetylsalicylate, naproxen, ibuprofen and etodolac. Previously, NSAIDs were considered as first-line drugs, however low effectiveness in prevention of damage progression and side effects at high doses such as nausea, abdominal pain, ulcers and gastrointestinal bleeding, limited the implementation of these drugs [68].

Surgery is the final treatment approach for RA therapy in cases when the aforementioned nonsurgical methods are not sufficiently effective, which are becoming less frequent. Nowadays, various types of surgery are being applied, among them are tenosynovectomy, radiosynovectomy, arthroscopy, osteotomy and joint replacement. The final goal of surgical management is to relieve pain and restore joint function ^[69][70][69,70]. Table 1 summarizes the current approaches for RA treatment with the route of administration, mechanism of action and major side effects.

Table 1.

Summary of current approaches for RA treatment.

Drug	Example	Administration/Dose	Mechanism of Action	Side Effects	Reference
Conventional Synthetic DMARDs	MTX	Orally or intravenous (IV) injection (15 mg), single subcutaneous (SC) or intramuscular (IM) injection (15–25 mg/week)	Impairs purine and pyrimidine metabolism, inhibits amino acid and polyamine synthesis	Skin cancer and gastrointestinal, infectious, pulmonary and hematologic side effects, bone marrow impairments	[27][28]	[27,28]
	Leflunomide	Orally (50 mg/week or 10 mg/day)	Inhibits dihydroorotate dehydrogenase enzyme leading to inhibition de novo synthesis of pyrimidine nucleotides	Dyspepsia, nausea, abdominal pain and oral ulceration	[71]	[72]
	Sulfasalazine	Orally (500 mg/daily or 1 g/day in 2 divided doses up to a maximum of 3 g/day in divided doses)	Suppresses the transcription of nuclear factor-κB (NF-κB) responsive pro-inflammatory genes including TNF-α	Nausea, vomiting, anorexia, dyspepsia, male infertility (reversible), headache and skin rash	[72]	[73]
	Hydroxychloroquine	Orally (400 mg/day over a 30-day period)	Increases pH within intracellular vacuoles and alters processes such as protein degradation by acidic hydrolases in the lysosome, assembly of macromolecules in the endosomes and post-translation modification of proteins in the Golgi apparatus	Retinal toxicity, neuromyotoxicity	[73][74]	[74,75]
Biologic DMARDs	Etanercept, Infliximab, Adalimumab, Golimumab, Certolizuma-bpegol	Etanercept—SC injection (50 mg/week or 25 mg/twice a week); Infliximab—SC injection (3–10 mg/kg every 4–8 weeks); Adalimumab—SC injection (25 mg/twice a week); Golimumab—SC injection (50mg/month); Certolizumab pegol—SC injection (400 mg at weeks 0, 2 and 4, followed by 200 mg every 2 weeks)	Blocks the biological activity of TNF	Infections, neurological diseases, development of multiple sclerosis and lymphomas	[75]	[76]
	Anakinra	SC injection (75–150 mg or 0.04–2 mg/kg)	Binds to IL-1 receptors	Opportunistic and latent infections	[76]	[77]
	Rituximab	IV injection (1 gm twice separated by 2 weeks) with MTX and IV corticosteroid premedication	Anti-CD20 monoclonal antibody	Hypogammaglobulinemia, rarely serious infectious events	[77]	[78]
	Abatacept	IV injection (2–10 mg/kg on days 1, 15 and 30, and then every 4 weeks)	Contains the domain of cytotoxic T lymphocyte–associated antigen 4 (CTLA-4), blocks interaction between DCs and T cells	Serious infections, increased risk of certain malignancies	[78]	[79]
	Tocilizumab	IV injection (8 mg/kg once every 4 weeks) or SC injection (162 mg/week)	Blocks IL-6 receptor	Serious infections, major adverse cardiovascular events, cancers, diverticular perforations, hepatic diseases, rarely lethal	[79]	[80]
	Secukinumab	SC injections (25–300 mg)	Primarily targets IL-17A	Nasopharyngitis or infections of the upper respiratory tract, mild-to-moderate candidiasis	[80]	[81]
	Brodalumab	SC injection (70–210 mg)	Prevents the nuclear factor kappa light chain enhancer of activated B cells, IL-6, IL-8, COX-2, MMPs and GM-CSF	Nasopharyngitis, upper respiratory tract infections, arthralgia, back pain, gastroenteritis, influenza, oropharyngeal pain, sinusitis	[81]	[82]
Targeted Synthetic DMARDs	Tofacitinib	Orally (5 mg/twice daily)	Blocks Janus kinases (JAK1 and JAK3)	Cardiovascular events, neutropenia and lymphopenia, risk of infection (viral reactivation, herpes virus reactivation, opportunistic infections)	[82]	[83]
	Baricitinib	Orally (4 mg/day or lower dosage 2 mg/day)	Inhibits JAK1/JAK2	Hyperlipidemia, viral reactivation, deep venous thrombosis and pulmonary embolism event	[83]	[84]
	Upadacitinib	Orally (15 mg/day or 30 mg/day)	Inhibits JAK1	Upper respiratory tract infection, nasopharyngitis, and urinary tract infections, gastrointestinal perforation	[84]	[85]
GCs	Dexame-thasone, be-tamethasone, triamcinolone, prednisone, prednisolone	The addition of GCs, to either standard DMARD monotherapy or combinations of synthetic DMARDs with low-dose GCs (< 7.5 mg/day) or high-dose GCs (up to 15 mg/day)	Directly activates or represses gene transcription	Ecchymosis, cushingoid features, parchment-like skin, leg edema, sleep disturbance, immunosuppression, weight gain, epistaxis, glaucoma, depression, hypertension, diabetes	[40][41][85]	[40,41,86]

All of the described therapeutic strategies aim to maintain disease remission or low progression as well as reduce the risk of treatment, and thus, have relative efficacy. About 20–30% of moderate-to-severe RA patients are unresponsive to current treatment strategies ^[86][71]. In this regard, stem cell-based therapy with its immunomodulatory and immunosuppressive properties represents a promising approach in RA treatment.

3. Mesenchymal Stem Cells in RA Treatment

MSCs are adult multipotent stem cells with fibroblast-like morphology. Due to their immunomodulatory properties, MSCs are being considered for the treatment of autoimmune diseases [87]. The original definition of human MSCs is based on the presence of fibroblast-like morphology, with their ability to adhere to culture plastic and to differentiate into tissues of mesodermal origin, such as osteoblasts, chondrocytes and adipocytes. Furthermore, MSCs express CD73, CD90 and CD105 cell surface markers, but not hematopoietic and endothelial markers (CD14, CD34, CD45 and HLA-DR) ^{[88][89][90][91]}[88,89,90,91]. The source of MSCs is a variety of tissues, including but not limited to, bone marrow (BM), gingiva, synovium, periosteum, adipose tissue (AT), dental pulp, umbilical cord (UC) and umbilical cord blood (UCB) ^{[92][93][94][95][96][97][98]}[92,93,94,95,96,97,98]. In addition to their ability to differentiate into multiple cell lines, MSCs are capable of modulating innate and adaptive immune responses by alleviating the proinflammatory phenotype, particularly, through decreasing populations of DCs, macrophages, NK cells, B and T cells, and by promoting anti-inflammatory phenotype ^{[11][99][100][101][102]}[11,99,100,101,102]. Depending on the environment, MSCs have the ability of polarizing and acquiring either pro-inflammatory (MSC1) or anti-inflammatory phenotypes (MSC2). In the presence of the inflammatory milieu (high levels of TNF-α and IFN-γ), which is generated by the immune cells, MSCs become activated and adopt an anti-inflammatory phenotype [11]. The mechanism of immune cell suppression by MSCs is mediated by secretion of a number of soluble factors, such as enzymes, cytokines and growth factors, including IDO, PGE2, NO, TGF-β1, HGF, hemoxygenase (HO), COX-2, IL-6 and IL-10. The IDO secretion is presumably induced by inflammatory cytokine IFN-γ [103]. The mechanism of action of IDO is mediated by conversion of an essential amino acid tryptophan to kynurenine, which impairs the synthesis of various cellular proteins and leads to the suppression of T cell proliferation. IDO is also considered to be involved in the generation of Tregs and tDCs induced by MSCs [104]. Moreover, factors produced by MSCs include nitric oxide synthase (iNOS), which induces the production of NO from macrophages thus inhibiting the proliferation, secretory and cytolytic functions of T cells. Both soluble factors function in the process of immunosuppression. However, it is demonstrated that iNOS mediates immunosuppression by mouse MSCs, while IDO plays a similar role in human MSCs [105].

Together with constitutive secretion of TGF-β by MSCs, the environment favors generation of Tregs ^{[13][106][107][108][109][110][111]}[13,106,107,108,109,110,111]. In the absence of an inflammatory environment (low levels of TNF-α and IFN-γ), MSCs may adopt a pro-inflammatory phenotype and enhance T cell responses by secreting chemokines (e.g, MIP-1a and MIP-1b, RANTES, CXCL9 and CXCL10) that recruit lymphocytes to the sites of inflammation; these chemokines bind to CCR5 and CXCR3 expressed on T cells. The levels of immune suppressive mediators, such as IDO and NO, are low when the pro-inflammatory phenotype is adopted ^[12][112][12,112].

The role of apoptotic MSCs for therapeutic applications have been recently investigated. A study by Galleu and colleagues demonstrated that infused MSCs undergo extensive caspase activation and apoptosis in the presence of cytotoxic cells, which is a requirement for their immunosuppressive function, in both preclinical and clinical studies [113]. The mechanism is explained by the engulfment of apoptotic MSCs by phagocytes, and the IDO production, which is ultimately necessary for mediating immunosuppression. Similar results were reported by another group. The MCSs effect is based on the hypoxia-induced activation of caspase 3-mediated apoptosis, recruitment of immune cells at the transplantation site and their further engulfment by locally circulating macrophages [114].

Thus, the mechanism of immunomodulation by MSCs is regulated by both cell-cell interactions and paracrine effect via the secretion of soluble factors. Considering the abovementioned immunomodulatory properties, MSCs are being widely investigated as a promising tool for the treatment of autoimmune diseases, including RA.