The Role of Cell Organelles in Rheumatoid Arthritis with Focus on Exosomes

What is deemed to culminate in auto-immune diseases is the inability of immune system to distinguish self-cells from non-self-ones. Recognizing the self-cells as foreigner, the immune system attacks and impairs self-molecules. Auto-immunity has been estimated to encompass wealthy nations which accounted for well over 25%, following a swift increase incidence therefrom [1, 2]. Such disorders are prevalent among people being at the age of between 20 and 40. Auto-immune disorders are typically chronic and incapacitating illnesses with a significant medical and financial burden [3,4,5].

In Rheumatoid Arthritis (RA), aggressive synovial hyperplasia – which destroy articular joints – is a significant hallmark. Several genetic, epigenetic, and environmental factors are held culprits for the initiation and progression of RA [6]. Immune and non-immune cells, non-genetic factors, inflammatory mediators are considered to involve in inflammatory processes which target cartilages and bones, resulting in a noticeable decline in the function of joints. Along with these components, variety range of vulnerable genes, namely human leukocyte antigen (HLA) class II, as well as more than 100 susceptible loci are responsible for reducing joints function. Even though M1 macrophages, T helper 1 (Th1), and Th17 cells have a key role to play in producing pro-inflammatory cytokines, such as Tumor necrosis factor (TNF), interferon gamma (IFNγ), interleukin (IL)-12, − 17, − 18, − 22, − 23, which contribute to the onset of auto-immune diseases, M2 macrophages have the ability to reduce the inflammation and to mitigate the condition via secretion of anti-inflammatory cytokines including IL-4, − 10, − 13, − 35 and transforming growth factor beta (TGF-β) leading [7].

Herein the role of cell organelle such as exosomes in rheumatoid pathogenesis and the therapeutic application of exosomes in RA will be discussed in detail.

Sundry Phases in RA Pathogenesis

Several aspects have been deemed to exacerbate arthritis, namely genetic, epigenetic, and environmental factors. Environmental risk factors which are included chemicals, smoking, and microorganisms can cause per se local inflammatory responses and immune system induction followed by epigenetic and post translation modifications (PTMs) of proteins [8].

Presentation of native or pseudo-native peptides (in breakdown of tolerance mechanisms) via dendritic cells culminate in T- and B-cells activation, thereafter it produces cytokines and autoantibodies. It was also pointed out that autoantibodies developed during the onset of clinical disease were able to recognize many neoepitopes on the process of epitope spreading [9].

Autoimmune responses to post-translationally both modified and unmodified self-antigens embark upon the disease, prior to emerging subclinical synovitis and clinical symptoms. Furthermore, autoantibodies produced during this preclinical process can cause both bone degradation and pain. Auto-antibodies are attached to various epitopes and create immunological complexes, resulting in synovial inflammation and arthritis [9, 10].

Many tissues are targeted in the RA cases and the main of which is the synovium. Inflammatory and joint-destructive substances are primarily stored in these cells. Auto-antibodies have an important role as mediator of joint inflammation and bone degradation to accelerate the inflammatory processes. Depending on the severity of the disease, auto-antibodies are found in 50–80% of RA patients [11, 12]. These antibodies could activate inflammatory effector pathways in chondrocytes and cartilages, leading to the release of extracellular matrix (ECM) components [13].

In this case, autoantibodies’ glycosylation is crucial. It has been reported that increased IgG-Fc sialylation has been associated to reduce inflammatory bone decay. However, decreased sialylation has been associated to RA and osteoclastogenesis. In short, the main causes of bone degradation are synovial inflammation, pro-inflammatory cytokines, autoantibodies, and receptor activator of nuclear factor B ligand (RANKL) [14]. In the situation in which self-tolerance is disrupted, immune and non-immune cells may be activated, thereby releasing inflammatory mediators. Noteworthy, preosteoclasts can be differentiated at the cartilage-bone interface into bone-resorbing osteoclasts as a result of fibroblasts expressing RANKL and macrophage colony-stimulating factor (M-CSF) [14]. It has been become a prevailing notion that T- and B-cells, signaling molecules, pro-inflammatory mediators, and synovium-specific targets ought to be considered as the novel therapeutic targets [10].

Role of Different Organelles in RAMitochondrial in RA

Mitochondrial malfunction has been shown in RA, neurological diseases, diabetes, cancer, and obesity. The linkage between Rheumatoid Arthritis and mitochondrial dysfunction has been shown by numerous studies. Nuclear DNA (nDNA) encodes some proteins that translocated to mitochondria. In the recent times, it has been reported that such proteins have a pivotal role to play in mitochondrial dysfunction, in particularly, in the cases of oxidative stress-related processes, apoptosis, and RA.

The provocation of internal nucleic acid sensors as well as the Toll-like receptor (TLR) 9 are induced by unmethylated CpG patterns in mitochondrial DNA (mtDNA). The immune response of mtDNA is increased by containing 8-oxo-guanine residues. These residues, albeit being far more common in mtDNA than in nDNA, are as a consequence of oxidative events which were caused by reactive oxygen species (ROS) [15]. ROS may arise in the case of RA synovial mitochondria as a result of both the pannus – which increases Adenosine triphosphate (ATP) demand and disrupting microvasculature – which leads to hypoxia through mtDNA, proteins, and lipids serving as initial targets for these free radicals. The pro-inflammatory HIF-1, NF-B, JAK-STAT, AP-1, and Notch pathways are also known to be activated by hypoxia and ROS [15].

In addition to aforementioned, the 1158 nDNA-encoded proteins can be translocated to mitochondria as well as involving in ROS production pathways [16]. In term of pathophysiology, the roles of such proteins in mitochondrial dysfunction and correlation thereof with ROS-mediated, hypoxia, ATP production, and inflammation in the case of RA is considered a vital subject to understand.

It is worth noticing that the apoptosis is important for synovial hyperplasia in RA to be controlled, as it can be triggered by both extrinsic and intrinsic processes. The intrinsic occurs in mitochondria caused by oxidative stress, whereas the extrinsic routs are inactive in the fibroblast-like synoviocytes (FLS) of RA patients. Notably, both mechanisms can lead to the activation of a protease cascade, known as caspases [17].

To recognize importance molecules in cytokine signaling in RA, protein-protein interaction (PPI) networks has been created by some researchers at the cellular level [18]. Importantly related regions, ego networks, and genes in PPI had also pointed out by studies that have link to RA and other illnesses. The PPI was also employed in another study to evaluate the efficacy of the anti-inflammatory medicines, Leflunomide, and Ligustrazine in the treatment of RA [19].

The existence of the electron transport system in mitochondrial has a key role in the modifications of mtDNA. In addition, the absence of histones in mtDNA makes them more vulnerable to ROS induced damages. There are two biochemical variables that influence into immunological capacity. Protein synthesis process in mitochondria is like that of bacteria which begins with a formylated amino acid. Formylated peptides can also stimulate neutrophil receptors, inducing activation and chemotaxis. The other mitochondrial protein – mitochondrial transcription factor A (TFAM) – is immunologically active. TFAM having similar structure to high-mobility group box 1 (HMGB1) can function as an alarmin and promotes inflammation like nucleus counterpart thereof [20].

Some chemicals may leave the mitochondria as permeability breaks down because of cell stress. Cytochrome c will elude the mitochondria’s sinking ship and interact with other molecules, causing apoptosis. In the case that of mtDNA is outside the mitochondria, it can activate the inflammasome by interacting with internal sensors. Owing to sever cell malfunction, all these mitochondrial products leave the cell, and also along with individual components which leak or release, entire mitochondria might depart from the cell with the payload of hazardous molecules. Following the stimulation of eosinophils, the mitochondrial catapult as an intelligent mechanism allows the entire mitochondria to be exteriorized [21].

Platelet activation is the epitome of presenting entire mitochondria in an extracellular position [22]. Microparticles comprised of mitochondria as well as whole mitochondria are found in the microparticles generated after platelet activation. The presence of entire mitochondria in the extracellular space is prominent, showing its similarity to bacteremia occurring during infection. Indeed, this demonstrates the contribution of mitochondria in shock and sterile inflammation [23]. Mitochondrial products having systemic and local functions in chronic autoimmune and inflammatory diseases are involved in the pathogenesis of disorders like RA by promoting synovitis via mtDNA in joints [24].

Terminology is affected by changing personality of mitochondria and the desire for becoming outright hazardous. Relying on the origin of functions, mitochondrial products differ from one another. For instance, products can be DAMPs (disease-associated molecular patterns) and PAMPs (pathogen-associated molecular patterns) if the activity are triggered by cell death and the same molecular structures, respectively [25].

To refrain conflict and prolonged disputes, it is better to use a neutral and non-committal word; to wit: MAMP (mitochondrial-associated molecular pattern) refers to a form of mitochondrial molecular pattern. The discovery of immunological potential of the mitochondrion promotes the increasing picture of molecules which are repurposed to enhance innate immunity [26].

Endoplasmic Reticulum in RA

The endoplasmic reticulum (ER) biosynthesizes both secretory and membrane proteins. The ER lumen provides a desired situation which is required for proper folding of membrane and secretory proteins. The homeostasis in the ER is preserved by the unfolded protein response (UPR), ER-associated degradation (ERAD), and a structured adaptive program. A variety of factors, such as altered cellular metabolism, mutations in substrate and route chaperones, and infection, are involved in predisposing proteins to misfolding [27, 28].

The initiation of a UPR signaling cascade throughout ER stress – not to mention it is caused by the aggregation of unfolded proteins – is done by the glucose regulated protein 78 kDa (GRP78) which has been known as BiP (binding immunoglobulin protein) [29]. Incidentally, three ER-localized protein sensors, namely double stranded RNA-dependent protein kinase RNA-like endoplasmic reticulum kinase (PERK), inositol-requiring transmembrane kinase-endoribonuclease-1 (IRE1), and activating transcription factor 6 (ATF6) contribute to the progression of the key UPR signaling cascade. The GRP78 impedes PERK, IRE1, and ATF6 to be activated in the resting state through merging into their N-termini. Upon being activated, the GRP78 may be attached to misfolded or unfolded proteins and liberate PERK, IRE1, and ATF6, inducing UPR signaling. The intrinsic ribonuclease activity of IRE1 promotes the formation of X-box binding protein-1 (XBP-1) – a transcription factor which escalates the expression of genes contributing to protein folding and degradation. Inhibition of the phosphorylation of initiation factor 2, nonetheless, can prevent general protein synthesis by PERK [30].

It is worth mentioning that the cell might lose the ability to rectify the protein folding deficiency and restore ER balance under long-term ER stress, thus the activation of cell death programs such as apoptosis and autophagy by the UPR. It has been shown that many diseases such as cancer, ischemia/reperfusion injury, neurological disorders hypoxia, cardiac disease, inflammatory bowel disease, infection, obstructive airway disease, and diabetes are associated with the ER stress response. The impaired ER stress, in turn, can lead to chronic autoimmune inflammatory disorders. The implication of a microarray study, moreover, revealed that the ER response can be escalated in skeletal muscle damage in the situation in which autoimmune myositis as the GRP78 expression is exerted in the muscle tissue of these patients [31].

The impact on RA of ER stress has remained a subject of intense debate. According to recent studies, a linkage between ER stress response and chronic autoimmune inflammation has been reported, whereby ER stress may induce or affect inflammatory disorders’ phenotype. Likewise, a study carried out to realize the impact of GRP78 on RA pathogenesis reached meaningful consequences. In the inflamed RA joints, some conditions are capable of increasing ER stress in both innate and adaptive immune cells; to wit: hypoxia, ROS, glucose deprivation, and pro-inflammatory cytokines. Inasmuch as the expression of GRP78 in RA-FLS is particularly upregulated in response to ER stress, such event enhances FLS survival and proliferation, hence synovial proliferation. The level of ER stress-mediated GRP78 in the ER lumen increased then can re-localize to cell surface from the ER, which in turn can be considered a target for Anti-citrullinated protein antibodies (ACPAs) and act as an auto-antigen for T- and B-cells [32]. Furthermore, extracellular GRP78 found highly in RA joints can promote the production of IL-17 and TNF in RA synovial mononuclear cells, and also it increases the growth of auto-reactive T-cells.

In addition, having bound to ACPA [33], citrullinated GRP78 on monocytes/macrophages escalates GRP78 expression in RA-FLS by promoting the production of pro-inflammatory cytokines, such as TNF α. This event per se can lead to amplifying the inflammatory cascade via promoting the formation of pannus [34]. Eventually, GRP78 promotes vascular endothelial growth factor (VEGF)-induced migration/chemotaxis and the endothelial cells proliferation as well as stimulating synovial angiogenesis, and also it has been considered as a vital ER chaperone [35, 36].

According to several studies carried out to ascertain other functions of GRP78, they pointed out that both cytoplasm and cell membrane are encompassed of GRP78, and also it plays a remarkable role in cell survival, metastasis, tumor angiogenesis, and resistance to chemotherapy. The discovery that GRP78 exists on FLS surface can pave the way to novel therapeutic means targeting the pathologic hallmarks of RA, synoviocyte proliferation, and endothelial cells [37].

The conjunction of toxin or apoptosis-inducing with the synthetic peptides having the potential to be blended with GRP78 such as WIFPWIQL peptide, may suppress synovial angiogenesis, proliferation, and pannus development [38]. It is important to note that since extracellular GRP78 induces T cell tolerance and rivals with membrane GRP78 to bind with the anti-GRP78 antibody, it can decrease RA activity.

Although RA-FLS studies, focusing mainly on changing occurred in cellular viability and possible repercussions for synovial hyperplasia thereof, it was myeloid-specific manipulation of UPR pathways that was shown to culminate in a remarkable decline in cytokine expression and in reducing K/BxN serum-induced arthritis. It was also pointed out that ER stress condition affect the RA-FLS to resist apoptosis, owing to increasing the rate of autophagy and the activation of proteasoms. Even so, our knowledge is limited of how ER stress has impact on FLS ability to influence synovial inflammation.

In recent times, a linkage between XBP1 splicing and TLR-mediated activation of RA-FLS has been reported [39]. Noteworthy, the presence of a significant ER stress signature is a distinguishing hallmark of RA synovium, as the IRE1α-XBP1 axis in RA synovial tissue is conducive to macrophage responding to TLR signaling in the condition being devoid of ER stress. Nonetheless, TLR stimulation increases cytokine and chemokine production in the cases of presence ER stress in stromal cells. Despite what has just been mentioned, how ER stress condition influences pathogenic processes has remained unknown [40].

Cytoskeleton in RA

The actin cytoskeleton fulfills many functions such as cellular homeostasis, modification of cell shape – migration, differentiation, and development thereof. It also has pivotal role in wound healing, polarity maintenance. To accomplish adequately such duties, it is imperative for every cell to coordinate its actin cytoskeleton with all external and intracellular. For instance, the number of both filamentous F-actin against monomeric G-actin and stress fibers are necessity to be controlled so as to movement. To this end, a complex intracellular protein network must be built, thereby allowing the cell to control actin polymerization, nucleation, depolymerisation, and actin organization relying on the cell’s needs [41].

Modulation of actin organization as well as controlling actin polymerization is of great importance. As a result, variety of signaling and/or actin-binding proteins can alter the actin cytoskeleton structure, leading to focal adhesion, stress fibres, lamellipodia, or even filopodia formation based on the needs of specific cells at the given time [41,42,43]. Notably, tiny GTPases of the Rho family proteins – Rho, Rac1, and Cdc – belonging to the Ras protein family and having been found in sundry isoforms, modulate the formation of projections in the membrane enclosing the cell. These proteins are able to bind with GTP and to impart an active GTP-bound form to an inactive GDP-bound form on a regular basis. The other function of such proteins was shown to interact with several effector molecules in the active state to deliver a specific signal. Rac1 and Cdc42 mediate the production of lamellipodia and filopodia if they be activated by growth factors and integrins, respectively [44, 45].

Nevertheless, it has been discovered that the activation of TNFα-induced NF-kB and cytokines secretion depends on the activation of RhoA in human cultured rheumatoid arthritis synovial fibroblasts (RASFs), which in turn this implies a crucial function of this GTPase in the arthritic inflammatory response. The implication of this issue is that the development of stress fibres can be exerted by Rho. Moreover, it has been pointed oud that the depletion of gelsolin – an actin binding protein involved in actin depolymerization by preventing F actin production – has exacerbated the illness in mice, resulting in the importance of gelsolin in regulating the actin cytoskeleton in RASFs of the RA cases [46, 47].

According to a recent research on cadherin-11-deficient mice, the importance of tissue remodeling in RA was highlighted. The implication of that was to realize an important role of RASFs in reducing the migration, invasion, and adhesion capabilities. Whereas the resistance to the K/B6N serum-transfer arthritis (STA) was shown to be accomplished by RASFs [48, 49].

In addition to aforementioned, the actin cytoskeleton has noticeable impact on cellular morphology and gene transcription, hence activating Mal – a modulator of gene expression. Following F-actin formation, Mal is separated from G actin, thenceforth it stimulates the serum response factor (SRF) as well as target genes which include genes controlling proliferation, cell growth, differentiation, and the actin cytoskeleton machinery. Interestingly, a dynamic relationship between actin cytoskeleton remodeling and SRF was found, as the ablation of the SRF can cause low F-actin levels and remarkable decline in migration and adhesion abilities in murine embryonic stem cells [50, 51]. Furthermore, increased F-actin synthesis may cause increased gene expressions which are mediated by SRF and NF-kB, leading to the maintenance of the disorganized actin cytoskeleton and finally to the generation of pro-inflammatory cytokines. All this event holds the arthritogenic response’s negative effects [50, 51].

More importantly, the actin cytoskeleton in numerous disease states, aside from RA, has the ability to maintain cell homeostasis and survival as well as playing significant role to keep going the function of many proteins being essential to fine-tune their organization. And also, a linkage was revealed between the failure in actin collection and polymerization in polymorph nuclear leucocytes with recessively inherited genetic condition known as neutrophil actin dysfunction. The actin polymerization is therefore important for neutrophil to move [52].

What has been found to be increased among patients suffering from RA is the serum amyloid level; this elevation results in synovial hyperplasia and angiogenesis. Actin and/or actin-binding protein-associated diseases have been linked to affect cancer cells, as they can affect tumor cells to progress and to metastasize [53]. Incidentally, various changes occurring during the metastatic process and tumor progression are as a result of deregulating the actin-binding proteins and actin cytoskeleton at either cellular matrix or cell–cell adhesion sites – not to mention such event happens in RA as well [52]. To clarify to understand by an example, diminishing expression of E-cadherin, deemed a cell–cell adhesion molecule, has caused poor prognosis and cancer progression. Deregulation of many ECM adhesion proteins, irrespective of having direct or indirect association with actin, play key roles in tumor progression and metastasis by either altering cell proliferation or generating anoikis – which is a type of apoptosis highlighted by the loss of ECM contact and adhesion [54, 55]. Notably, it has been recommended that some of which be considered as potential targets for novel anticancer therapeutic methods [43]. To achieve this goal that the actin cytoskeleton can be considered as novel therapeutic method, the suppression of signaling inducing actin polymerization by either short peptide compounds or direct interference with their gene expression can be an effective way.

Antikeratin antibodies have been found among nearly 68% of patients suffering from rheumatoid arthritis hand abnormalities, reaching a prevailing notion that there is an important linkage between Antikeratin antibodies and RA [56]. Since Antikeratin antibodies act against citrulline, actin and myosin may become a target for anti-cytoskeletal antibodies upon citrullination. Some proteins citrullinated can induce this process, too. In short, patients afflicted by rheumatoid arthritis were shown to have higher amount of autoantibodies acting opposite to cytoskeletal antigens, particularly the myofibrillar proteins actin and myosin [57, 58].

Exosome and Extracellular Vesicles in RA

Exosomes are nanoscale cell-derived extracellular vesicles (EVs) with 30–100 nm in diameters [59]. They can be generated by the reverse budding of multivesicular bodies and extricated when they fuse with the plasma membrane. Exosomes were originally assumed to be used for removing unwanted proteins from cells and were named “trash cans” [60]. Later, they were used to deliver bioactive compounds, such as proteins and microRNAs, in intercellular communications, called cytosol gulps [61].

Exosomes fulfills various function as well as having noticeable roles in sundry processes irrespective of type of cells. The most important of such functions has been noticed is to transmit bioactive molecules among cells. Along whit transmitting, some conditions such as transmissible spongiform encephalopathies, Alzheimer, Parkinson, and amyotrophic lateral sclerosis can be affected by exosomes [62,63,64]. Exosomes have been reported to be shed by both immune cells, namely mesenchymal stem cells (MSCs), mast cells, lymphocytes, and dendritic cells, and even notably tumor cells [65, 66].

Depending on conditions and correlations, exosomes can deliver different substances. As an example, exosomes contained the TCR/CD3/zeta complex and major histocompatibility complex class II (MHC II)-peptide can be shown in the situation in which T-cells correlate with antigen presenting cells (APCs) [67]. Additionally, MSCs increase remarkably releasing exosomes bearing anti-inflammatory molecules which are imperative for polarizing macrophages into the M2 phenotype in the case of hypoxia preconditioning and in the presence of lipopolysaccharide [68].

Dependent and independent fusions have been considered as two different methods accomplished by exosomes to influence into nearby cells. Fusions per se are divided into two categories: 1) direct fusion pertaining to plasma membrane of the recipient cell – which relies on receptor-ligand interactions, 2) Back fusion relating to the vesicles are undergone endocytosis by recipient cell and hence the inclusion of the vesicles into the endosome’s membrane. However, the inverse function is shown in the fusions encompassed independent method, as the interaction between vesicles and recipient cells is incident. Function of delivering MHC-peptide having link to the T-cell receptor (TCR) on T-cells fulfilled by exosomes is the quintessential of such fusion [69, 70]. To identify exosomes, it can be done by different aspects, such as exosome-specific membrane proteins, cell types, condition-specific proteins, genetic materials, mRNA, and miRNA.

Long noncoding RNAs (lncRNAs) has been become a demand subject to investigate since it has impact on embryonic differentiation, pluripotency, and development. It was also reported to play key roles in variety of illnesses [71]. Serum exosomal Hotair – HOX transcript antisense RNA found as the first lncRNA – was shown to correlate highly with clinical features of laryngeal squamous cell carcinoma (LSCC), particularly in the situation in which the level of its serum expression is evaluated. That is why it can be used as a biomarker to monitor LSCC and be considered as a prediction tool for patients afflicted by LSCC. What is worth mentioning is that it has been discovered among the exosomes of patients suffering from RA in the case of high expression level of Hotair, culminating in promoting macrophage migration [72]. Conversely, the amount of Hotair among some disorders including differentiated osteoclasts and rheumatoid synoviocytes has been shown negligible. This issue induces the expression of Hotair, thereby much lower level of matrix metalloproteinase (MMP)-2 and MMP-13 as well. Although more researches is imperative to reach a prevailing notion in terms of making use of exosome-derived Hotair so as to sundry subjects, it can be deemed as a potent biomarker to diagnose RA [73].

Mechanism of Exosome Function

Exosomes are thought to be vehicles delivering immunosuppressive substances generated from their parents’ dendritic cells (DCs), even though the precise functional

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