**2. Molecular and Cellular Mechanisms of Systemic Arthritis**

The pathogenesis of systemic arthritis manifested in childhood and adulthood is multifactorial, unclear, and very complex, in which the innate immunity plays an important role by activating neutrophils and macrophages, as well as the adaptive immunity, by increasing the percentage of pro-inflammatory cytokines: interleukin (IL)-1β, IL-6, IL-18, and interferon gamma (IFN)–γ [30–34].

sJIA accounts for about 10% of all forms of juvenile arthritis and is a chronic disease that results in significant morbidity and mortality in children [22,35].

The most significant manifestation of systemic arthritis is its association with macrophage activation syndrome, a secondary disorder of excessive, uncontrolled activation and non-malignant proliferation of T lymphocytes and macrophages, with a state of hypercitokinemia, on which the clinical–biological signs depend [36–38].

The underlying cause of the occurrence of chronic rheumatism in the JIA subtypes, including sJIA, is largely unknown. A current concept would be that triggering manifestations in sJIA would be due to an infectious aggression with an inappropriate immune response due to a genetic or acquired immune defect [33].

More and more studies have shown that in the pathogenesis of sJIA, the innate immune system is more involved, compared to the adaptive one [31,39,40].

Biological studies for sJIA describe a polymorphism of disease-promoting elements encoded by tumor necrosis factor alpha (TNFα), IL-6, IL-10, macrophage migration inhibitory factor (MIF), and IL-1 family (in particular, IL1A, IL1RN, IL1R2) [41–44].

More and more clinical and biological as well as translational research draw attention to the particularly important role of IL-1β, IL-6, and IL-18 in the complexity of disease manifestation and the limited role of TNF-α, as well as the relative absence of induced chemokines by interferon gamma (IFN-γ), IFN-γ-inducible protein 10 (IP-10, CXCL10), MIG, and I-TAC [31,40,45].

The IL-1 superfamily contains 11 cytokines, from which IL-1α and IL-1β have the most powerful pro-inflammatory effect. They have a natural antagonist IL-1Ra (IL-1 receptor antagonist), which is an endogenous inhibitor of IL-1 and works as a competitive inhibitor of IL-1 binding to IL-1R1. It is produced by inflamed synovial macrophages and initiates inflammatory responses [46].

IL-1 has a biphasic role in implicating innate immune mechanisms, but also in adaptive ones in triggering sJIA. At the onset of the disease, the disruption of immunity induced by IL-1 induces clinical manifestations of fever, rash, and early synovitis. Then, IL-1 intervenes in the mechanisms of adaptive regulation by activating and promoting the differentiation of T lymphocytes in Th17 cells with a pro-inflammatory role and by inhibiting the activity of T-regulatory cells [47].

Certain evidence of IL-1 involvement in the pathogenesis of sJIA is given by the successful treatment with IL-1 inhibitors, such as the biological agent anakinra, a soluble IL-1 receptor antagonist (IL-1Ra) that is similar to IL-1Ra, which has increased levels during the active disease observed also in polyarticular JIA. At the same time, with particularly good clinical results for anti-IL-1 therapy, the normalization of genes expressed by peripheral blood mononuclear cells (PBMCs) was observed [48–50].

Since the response to the IL-1β antagonist therapy of the anakinra product is limited by the large number of IL-1β receptors expressed on several different cell types and the rapid excretion of IL-1RA by healthy kidneys, two drugs (rilonacept and canakinumab) have appeared much more efficient [51].

However, not all patients with sJIA respond well to anti-IL-1 therapy. Clinical–biological research has shown that age at onset of disease, duration, number of active joints, polymorphonuclear, and serum ferritin levels can predict anti-IL-1 response. Elevated ferritin in patients' serum can be a valuable biological signal and parameter, which will guide us to explore macrophage activation syndrome (MAS), which can be effectively treated with anti-IL-1 agents. So far, the laboratory data have not revealed the presence of antibodies, and there is evidence that sJIA is actually a form of autoinflammatory disease, and not autoimmune [52].

The importance of IL-6 in the pathogenesis of sJIA has been demonstrated by correlating the serum and synovial concentration with the severe joint manifestations and the maximum level of fever [53].

Pro-inflammatory cytokines, such as IL-1, IL-6, and TNF-α, by stimulating similar receptors, induce IL-6 production in lymphocytes, macrophages, and synovial cells [54].

IL-6 has various functions in the pathogenesis of sJIA in children and rheumatoid arthritis in adults, in the sense that it induces an acute phase response as well as activates immune reactions and hematopoiesis.

The released IL-6 will induce the production of acute phase proteins (C-reactive protein and fibrinogen), which are known as biological markers of inflammation and the differentiation of naive T cells into Th17 cells [54,55].

The imbalance between Th17/Treg, where Th17 is activated significantly more than Treg, has a disastrous effect on RA development [56].

As IL-10 is a cytokine that would play a key anti-inflammatory role in the prevention of immune cascades from immune-mediated inflammatory diseases, conflicting results are reported in the literature regarding the poor involvement of this cytokine and the occurrence of manifestations in sJIA [43,44,57].

In a study published by Imbrechts et al., experimental evidence was provided on a mouse model that there would be a relationship between IL-10 insufficient production and its consequence in the innate cellular immune response from sJIA pathogenesis [58].

Both sJIA and macrophage activation syndrome are triggered by a cascade discharge of some cytokines such as interleukin 1β, IL-6 and IL-18.

To date, the exact role of interferon-gamma (IFN-γ), a cytokine with pro- and anti-inflammatory properties is being intensively investigated along with the role of NK cells providing IFN-γ [59].

Put K. found in its published PhD thesis on mice models that "the inflammatory environment in sJIA affects NK cells, causing an inflammatory transcriptional profile in these cells"; although there are very high plasma levels of IL-18, they do not influence NK cells, which causes IFN-y production to be low. Therefore, NK and IFN-y cells should be considered as limiting factors in sJIA pathogenesis [60].

Despite all previous knowledge that IL-18 is commonly recognized as the major inducer of IFN-y synthesis in NK cells, the paper published by Put K. et al. in patients with active systemic JIA shows that despite high plasma levels of IL-18, IFN-y levels remained low. In contrast, gene expression profiling was altered by the increased expression of innate genes, including TLR4 (Toll-Like Receptor 4) and S100A9 (S100 calcium-binding protein A9), and the decreased expression of immunity-regulating genes, such as IL-10RA (interleukin 10 receptor, alpha) and GZMK (granzyme K), as compared to cells from healthy controls. From these studies, it is believed that subtle defects in the pathways associated with NK cells, such as granzyme K expression and IFN-γ production determined by IL-18, may contribute to the immune aggregation of this disease [61].

#### *Macrophages Activation Syndrome*

Macrophage activation syndrome (MAS) or hemophagocytic syndrome is a complication of Still disease in children and adults, which can be life-threatening and is considered a subset of hemophagocytic lymphohistiocytosis (HLH) [62,63].

Macrophage activation syndrome generally has an unknown incidence because some forms are expressed by mild subclinical signs, but 10% of patients with sJIA could have a serious, potentially lethal complication [64].

From the biological point of view, MAS is expressed by a defect of the cytolytic pathways with an uncontrolled proliferation of cytotoxic cells and a hypersecretion of pro-inflammatory cytokines—that is, an increase in hematophagocytic T lymphocytes and macrophages that induce a cytokine storm with severe multiorgan injury [65,66].

In MAS, NK dysfunctions, mutations of the UNC13D, PRF1, STXBP2, and RAB27 genes, TLR-9 receptor dysfunction of IFN-γ, and activation pathways of IL-10 and IL-18 were observed [67–69].

Clinical symptoms include fever (96% of cases, often persistent), hepatomegaly (70%), splenomegaly (58%), and lymphadenopathy (51%). In 35% of cases, the neurological manifestations could be convulsions, drowsiness, irritability, confusion, headache, and coma. There may also be cardiac involvement even with pericarditis, pulmonary pleural effusion, hematuria, proteinuria, and signs of renal failure. Hemorrhagic manifestations range from purple rash, ecchymoses, gingival or gastrointestinal bleeding, and disseminated intravascular coagulation. MAS laboratory data include decreased ESR, WBC, platelet count and serum fibrinogen; as well as high and extremely high levels of ferritin, D-dimers, liver enzymes, lactate dehydrogenase, triglycerides, with the prolongation of prothrombin time (PT) and partial thromboplastin time (aPTT) [70–72].

Today, there are guidelines with diagnostic criteria for HLH, MAS associated with sJIA, or MAS associated with systemic lupus erythematosus. Selecting the right diagnostic criteria is essential for successful therapy [62].

#### **3. Comparative Pathogenesis of Rheumatoid Arthritis in Adults and Children**

The pathogenesis of RA and JIA is not yet very well known, although there is strong evidence that it involves the components of the immune system, especially T and B lymphocytes, as well as the antibodies and cytokines resulting from this immune conflict [7,73].

As is well known, the immune system has two main branches: the innate components and the adaptive immunity. The cells and receptors of the innate immune system play an extremely important role in rapidly recognizing the foreign infectious agent and initiating a defense response, which is known as pro-inflammatory [74].

In triggering an inflammatory action, innate immune cells—neutrophils, macrophages, monocytes, natural killer cells, dendritic cells (DC) and so on, playing the role of stopping the inflammatory process (infectious)—will inform, initiate, and direct the phenomena of the proliferation and differentiation of adaptive immune cells [74].

In response to the inflammatory aggression of B cells, the β- and γδ-selected T cells from the branch of innate immunity will be stimulated to proliferate and differentiate into cells specific to the functions appropriate to the immunological challenge, and they will eventually die and leave subsets of cells with memory. In rheumatoid arthritis, the activation of a naive T cell departing from the thymus to the lymphoid organs involves coordinated interactions between a number of molecules on the surface of this cell and an antigen-presenting cell (APC), that is, that carries an antigenic peptide derived from the infectious agent noncovalently linked to a major histocompatibility complex (MHC) class I or class II molecule.

When the APC cell is activated, various costimulatory ligands are expressed, allowing the activation, proliferation, and differentiation of T cells [74,75].

The T cells will express a series of inhibitory receptors for a fine regulation of the response appropriate to the inflammatory environment where they were being stimulated. Inhibitory receptors can act in two directions: to limit the costimulatory signaling, as well as to temporarily bind the costimulatory molecule [74].

At the synovial level, as result of inflammation, the differentiation of naive T cells in Th17 cells will occur. It was believed that this immune pathology would be mediated by Th1 cells (the first objectified), but today, the research has evolved, and it has been discovered that, in fact, Th17 cells are considered responsible for the pathogenesis in rheumatoid arthritis [76,77].

Current studies demonstrate that synovial fibroblasts and activated immune cells are directly involved in the production and release of many pro-inflammatory cytokines that play a crucial role in the development and progression of RA [78].

In fact, the characteristic inflammatory process in RA is achieved by the abundance of inflammatory-promoting cytokines, in counterbalance with inhibitory cytokines, intercellular communication, immune responses, and boosting cell movement to territories of inflammatory, infectious, or post-traumatic conflict.

In RA, the cytokines of the immune network are classified into four groups: pro-inflammatory cytokines, inflammatory cytokines in the joints, anti-inflammatory cytokines, and natural cytokine antagonists [79].

After the onset of initial stimuli, the cytokines play an important role in communicating with the components of the immune system at each stage of the pathophysiology of RA.

The release of cytokines, particularly the TNF-α, IL-6, and IL-1, promotes the synovial inflammatory process.

IL-6 binds to cells via a specific receptor complex involving two proteins, IL-6 receptor α and gp130, in order to transmit information. IL-6 receptor α exists into two forms: a transmembrane IL-6 receptor α, or mIL-6R (membrane-bound form of IL-6R), and a soluble IL-6 receptor α, sIL-6R. After IL-6 binds to any IL-6 receptor, the complex formed will induce gp130 activation. All IL-6-type cytokines signal through the gp130/JAK/STAT pathway. The binding of IL-6 to mIL-6R induces anti-inflammatory classic signaling, whereas the binding of IL-6 to sIL-6R induces pro-inflammatory trans-signaling [80–82].

Recent clinical studies have shown that patients with RA (adults) or active polyarticular sJIA (children), who did not respond adequately to MTX (methotrexate) and TNF-alpha inhibitors, received an IL-6 inhibitor, for example, tocilizumab in children and sarilumab (in adults), which are biologics that can be more effective [83,84].

Among the pro-inflammatory cytokines at the synovial level, TNF-α is a pleiotropic cytokine produced by several cell types, such as T and B cells, but also by innate immune cells (dendritic, monocyte, neutrophil, mast cells) and has a very important role, because it participates as the main mediator in regulating and training other factors [85,86].

In addition to this role, it is known that TNF-α is associated with bone and cartilage destruction by activating chondrocytes and osteoclasts [87].

TNF-α induces the synthesis and secretion of MMPs (matrix metalloproteinases), which in turn affect the chemokine and cytokine action of MMP-2, MMP-3, MMP-7, and MMP-9, which release TGF beta (Transforming Growth Factor) from the matrix, thus enabling its activation [88].

Therefore, anti-TNF biological therapy has been considered a remarkable breakthrough in the treatment of chronic autoimmune diseases, such as RA and JIA [85].

The interleukin (IL)-1 (family) together with its members (IL-33, IL-36α, β, γ, IL-37, and IL-38), IL-6, and IL-12 superfamilies (IL-27, IL-35) together with the other key cytokines (IL-15, IL-16, IL-17 family IL-17A, IL-17B, IL-17C), the recently cloned cytokine IL-18, IL-32, IL-34, and interferon (IFN)-y, the granulocyte macrophage colony-stimulating factor, are detected in a high concentration in the synovial fluid, but also in the patient's serum, thus leading to the process of local joint destruction and systemic effects in the rheumatoid arthritis patient [79,89].

More explicitly, IL-1 has 11 pro-inflammatory and anti-inflammatory members, which are chronologically numbered based on their discovery, from the IL-1 first family member 1 (IL-1F1) to IL-1F11. More commonly, they are also known as receptor antagonist IL-1α, IL-1β, IL-1 (IL-1Ra), IL-18, IL-33, IL-36α, IL-36β, IL-36γ, IL-36Ra, IL-37, and IL-38 [90].

IL-33 has been detected in high serum concentrations in adult patients with rheumatoid arthritis, in contrast to those with osteoarthritis (OA) and psoriatic arthritis (PsA) and was associated with bone erosion and cardiovascular pathology, as a predictive factor for the evolution of atherosclerotic

plaque [89]; however, the results are contradictory for its real role in the pathogenesis of RA, and as a consequence, specific drugs are not yet available [91–94].

Interleukin (IL)-17A is a pro-inflammatory cytokine that participates in the development of several autoimmune and inflammatory diseases [95].

IL-17A has a direct influence on the early pathogenesis and chronic stages of synovitis in rheumatoid and psoriatic arthritis, through systemic, but also local effects on keratinocytes [96].

The synovial membrane in RA is modified by inflammatory factors in the sense of the appearance of a local infiltrate of immune cells, hyperplasia, and angiogenesis tissue [97,98].

It has been shown that in the synovial lymphocyte infiltration and in the hyperplastic mucosa of RA, there are cells producing IL-17A and IL-17F; at the same time, there is a recruitment of Th17 cells that will interact with local cells and perpetuate chronic inflammation [99].

IL-17 is directly involved in the stimulation of vascular endothelial growth factor production in synovial fibroblasts, angiogenesis, and synovial pannus development [100,101].

The interaction between Th17 cells and synoviocytes is crucial, because as a result of this cooperation, IL-17 will be massively released [96].

TNF-alpha supports the effect of human IL-17A for the action of increasing the secretion of IL-6 and IL-8 from rheumatoid synoviocytes and vice versa, IL-17A and IL-17F induce TNFα receptor II expression and production [96,102,103].

Another particularly important role of IL-17 is to promote the expression of nuclear factor kappa-B (NF-κB) ligand receptor activator (RANKL) on osteoblasts and synoviocytes and to activate RANK signaling in osteoclasts [104–106].

Since 1999, it is known that IL-17 from human T cells activated in synovial tissues of patients with rheumatoid arthritis is a potent stimulator of osteoclastogenesis [107] and ultimately, the destruction of the bone [96].

The pro-inflammatory cytokines are also responsible for the synthesis of chemokines from MMPs, inducible nitric-oxide synthase, osteoclasts differentiation, and an increased expression of cell adhesion molecules. Disruption of MMP activity can lead to tissue degradation associated with inflammation in rheumatoid arthritis. Several inhibitors capable of modifying MMP activity are approved today, but unfortunately, they are associated with undesirable side effects [88].

Phytochemicals such as flavonoids, glycosides, lignans, and alkaloids are valuable natural sources for the development of new drugs with efficacy and safety in inhibiting upstream signaling molecules involved in MMP expression [88].

Helper T cells are deeply involved in the pathogenesis of autoimmune diseases, including RA; for example, it has been shown recently that Th17 can move into a "non-classical" class of Th1, with higher pathogenic activity, which is a phenomenon that further complicated the explanation of the pathogenic mechanisms of RA [108].

The same study has shown in patients with early-onset RA but without medication a higher ratio of Th17-derived Th1 cells comparatively to CD161 + Th17 cells, and an inverse correlation between interferon-γ (IFNγ) + Th17 cells, comparatively with the anti-CCP antibodies levels [108].

Today, it is known that Th17 produces the cytokine IL-17 [54], which activates inflammation by stimulating immune cells and at the same time activates osteoclasts by inducing kappa B ligand nuclear factor activator receptor (RANKL) in synovial fibroblasts. This fact opens new horizons for Th17-targeted therapies in order to stop the bone destruction associated with T cell activation [109].

At the same time, Foxp3 is essential for the suppressive function of Treg cells, and as a specific marker of Th17 cells, it accelerates osteoclasts differentiation. In RA, Foxp3(+)CD4(+) T cells are subjected to conversion into TH17 cells, which is mediated by synovial fibroblast-derived IL-6 [110], and meanwhile, IFN-gamma cytokines, IL-4, and cytotoxic T lymphocyte-associated protein 4 (CTLA-4), produced by Th1, Th2, and respectively, Treg, regulate osteoclast differentiation.

In RA, there is an imbalance in the Th17/Treg ratio, where Th17 is activated much more than Treg [111].

It is speculated that TNF inhibitors used in RA therapy reduce the passage of Th17 cells to non-classical Th1 cells, as well as direct inhibit the TNFα [85,108].

Inflammatory synovitis both in RA and JIA provides the image of an imbalance between pro-inflammatory and anti-inflammatory cytokines [IL-10, IL-11, and IL-13], which are insufficient to counterbalance the intensely active inflammatory process. Bone destruction in RA is caused by the effects of osteoclasts and not by the invasion of inflammatory factors directly into the synovium [112].

Synovial inflammatory cytokines [TNFα, IL-1, IL-6, and IL-17] promote excessive synthesis of (RANK)/RANKL (receptor activator of nuclear factor kappa-B ligand) on the membrane of synovial fibroblasts and/or osteoblasts and their differentiation [113,114].

Cartilage destruction is caused by MMPs or ADAMTS (a disinterring and metalloproteinase with thrombospondin motifs) that are produced by chondrocytes, synovial fibroblasts, and synovial macrophages. The JAK/STAT pathway is another signaling pathway for various cytokines and growth factors involved in the pathogenesis of rheumatoid arthritis. JAK is a tyrosine kinase receptor that mediates intracellular signaling through a transcription factor, STAT [115].

Currently, there is already experience with inhibitory drugs for the JAK receptor family; thus, tofacitinib for JAK1, JAK2, JAK3, and Tyk2; and baricitinib, which selectively act on JAK1 and JAK2 and are used in RA therapy [116,117].

Forty years after the discovery of IL-1, the "triggering agent" of the molecular and cellular mechanisms of the appearance and development of RA is constantly being sought as another fascinating field of intense investigation. In recent years, the range of pro- and anti-inflammatory cytokines has expanded rapidly with the identification of new members, and it has been proven to be involved to varying degrees in the pathogenesis of RA. Based on this knowledge, the therapeutic arsenal for RA patients includes monoclonal antibodies, fusion proteins, or antagonists against these molecules [89].

The treatment of rheumatoid arthritis in children and adults first benefits from methotrexate as "gold standard therapy", and if patients do not respond or experience complications and/or adverse reactions, TNF inhibitors will be given: infliximab, etanercept, adalimumab, golimumab, and certolizumab pegol (for adults). While TNF, IL-6, and JAK inhibitors directly regulate cytokine generation and bioactivity, a new biological product, abatacept (as a selective costimulation modulator), has been shown to inhibit T-cell activation by binding to CD80 and CD86, thereby blocking interaction with CD28. This results in the inhibition of autoimmune T-cell activation that is implicated in the pathogenesis of rheumatoid arthritis [118–120].

Although there are more and more biological agents with different mechanisms of action for the treatment of rheumatoid arthritis in children and adults, the results are not as we expected, because there are partial responses or non-responsive patients to these compounds, high therapeutic costs, side effects, and so on; therefore, we must turn our attention to other therapeutic modalities to induce disease remission.
