The role of bacterial infections in rheumatoid arthritis development and novel therapeutic interventions: Focus on oral infections

Abstract Background Rheumatoid arthritis (RA) represents a primary public health challenge, which is a major source of pain, disability, and socioeconomic effects worldwide. Several factors contribute to its pathogenesis. Infections are an important concern in RA patients, which play a key role in mortality risk. Despite major advances in the clinical treatment of RA, long‐term use of disease‐modifying anti‐rheumatic drugs can cause serious adverse effects. Therefore, effective strategies for developing novel prevention and RA‐modifying therapeutic interventions are sorely needed. Objective This review investigates the available evidence on the interplay between various bacterial infections, particularly oral infections and RA, and focuses on some potential interventions such as probiotics, photodynamic therapy, nanotechnology, and siRNA that can have therapeutic effects.

of the immune system, potentially triggering several immune processes. 10 Bacteria-derived lipopolysaccharide (LPS) could spread into the blood and be transported to the joints. Hence, LPS could stimulate innate immune receptors in the synovium, cartilage, and bone. 11 The development of biofilms is considered to be a driving force behind persistent infections. LPS has a significant role in biofilm formation. 12 Moreover, endogenous and microbial ligands can activate toll-like receptors (TLRs) and trigger an immune response in patients' derived cells. In particular, bacterial peptidoglycan and LPS induced the expression of chemokines IL-6 and (C-X-C motif) ligand 8 (CXCL8), via TLR-2 binding, in RA synovial fibroblast. ILs are a key element in infiltrating and maintaining the inflammatory cells within the synovium membrane. 13 Furthermore, macrophages with a high-level expression of TLR-2 resulted in an improper response to bacterial cell wall peptidoglycan. 14 Persuasive research has suggested a possible link between the immune response against oral infections and the development of RA through the production of enzymes by oral pathogens. 6,[15][16][17] Pathobiology of the disease itself, immunocompromising comorbidities, lifestyle, and immunosuppressive drugs appear to play an important role in creating the conditions for the development of this correlation as well. 18 Qiao et al. reported that patients infected by periodontal pathogens had a 69% greater risk for RA than individuals without periodontal disease. 19 The utilization of disease-modifying antirheumatic drugs (DMARDs) like methotrexate (MTX) is applied as first-line therapy for RA, but long-term use of MTX could not continue because of adverse effects and drug resistance. 20,21 To overcome the side effects, many researchers have investigated techniques to develop targeted delivery strategies. However, intra-articular injection provides the best-targeted therapy, frequent joint needling is usually necessary. Hence, this treatment choice might intensify the risk of infection. Also, RA involves all joints throughout the whole body.
Therefore, local administration of inflamed joints is not a suitable option. 22 The new approach for tissue-specific delivery of drugs, as well as enhanced drug bioavailability in the inflamed synovium while reducing off-target unwanted adverse effects, are very favorable. 23 A detailed understanding of the pathogenesis of RA will help us to develop complementary therapies such as probiotics, photodynamic therapy (PDT), nanostructures, and siRNA against RA in the pharmaceutical market.
This review aims to achieve a comprehensive understanding of bacterial populations in the oral cavity, which may support the idea that oral infections are a main modifiable risk factor for lifethreatening diseases. Furthermore, the new therapeutic options will also be discussed how targeting RA.

| R A IMMUN OPA THO G EN E S IS
RA is a chronic inflammatory disease involving impaired function of both the innate and adaptive immune systems. 24 Macrophages, neutrophils, mast cells, and natural killer (NK) cells are part of the innate immune system involved in the development of the inflammatory process in the joint. Antigen-presenting cells (APCs), such as macrophages, and dendritic cells (DCs), stimulate inflammation and release pro-inflammatory products, such as tumor necrosis factor-alpha (TNFα), cytokines interleukin-1B, 6, 18, 23, reactive oxygen species (ROS), and matrix-degrading enzymes involved in cartilage and bone destruction. 25 Especially TNFα is produced during the inflammatory process, mainly by activated monocytes/ macrophages. 26 Neutrophils have been known as a key contributing factor in the pathogenesis of RA. They increase inflammatory activity and tissue destruction by releasing pro-inflammatory cytokines, enhanced oxidative stress, granules containing destructive enzymes, and release of neutrophil extracellular trap (NET) and leading to cartilage and bone destruction. 24 Furthermore, TLRs activation plays a major role in RA pathogenesis. In RA patients, the aberrant activation of TLRs renders chronic inflammatory processes. 14 The TLR system recognizes LPS (TLR4), peptidoglycans and lipoteichoic acid (TLR2), and unmethylated CpG DNA (TLR9) from bacteria. 27 In addition, macrophages express TLR-2, resulting in an improper response to the bacterial peptidoglycan. 14 Many of these observations could be explained in RA patients, with upregulated responses to TLR-2 and TLR-4 ligands of blood monocytes and synovial macrophages. 28 Additionally, type 17 helper T-cells (Th17) have a crucial role in RA pathogenesis. IL-17A cytokines can also mediate fibroblast-like synoviocytes (FLS) and osteoclast activation, recruitment, and activation of neutrophils, synovial macrophages to secrete osteoclastogenic factors such as TNFα and IL1β. 29

| The ROLE OF PERI ODONTAL INFEC TI ON S IN R A
Periodontitis as a common infectious disease has attracted much attention in the field of public health. Periodontitis leads to progressive damage such as gingival recession, alveolar bone resorption, progressive resorption of periodontal supporting tissues, and eventual tooth loss. 30 A recent meta-analysis found that patients with periodontitis had a 69% greater risk of RA compared to healthy groups (OR = 1.70, 95%CI: 0.75-3.85, p < 0.001). 19 Porphyromonas gingivalis is a major periodontal pathogen that may be an important threat due to disturbing host immune homeostasis. LPS, gingipains, collagenase, proteases, fimbriae, lectins, capsule, and superoxide dismutase (SOD) are among the strategies employed by this pathogen to increase bacterial colonization and destroy host periodontal structures. In addition, virulence factors enhance the adhesion of P. gingivalis with other bacteria and biofilm build-up. Bacterial virulence factors also modulate or interfere with inflammatory response and disrupt the host immune response to escape bacterial clearance. 31,32 P. gingivalis expresses the citrullinating enzyme peptidyl arginine deiminase (PAD). Citrullination, a post-translational modification, leads to loss of tolerance to selfproteins in genetically susceptible individuals, inducing an immune response driving RA onset. Therefore, P. gingivalis infection causing protein citrullination could induce the generation of ACPAs and subsequent RA development. The presence of ACPA has been related to anti-P. gingivalis titres in RA patients. 33 Interestingly, this pathogen may alter the TLR response, subvert IL-8, or alter the complement cascade. 34 Neutrophils as the primary effectors of the innate immune system, immobilize and kill a broad range of microbes via NET formation. NETs are networks composed of chromatin, intracellular granules, and antimicrobial peptides with high bactericidal potential that form an extracellular matrix-like structure. 35 Although the antimicrobial defense structures of NETs are beneficial, accumulated NETs or delayed clearance of NETs might intensify the tissue damage and represent a source of autoantigens; indeed, ACPA-positive RA reacts strongly with histones found in NETs. 36 The potential immunogenic role of NETs is associated with the PAD4 activity, which arises the citrullination of histones and Eno, leading to a breakdown of immune tolerance to citrullinated epitopes. 37 Additionally, P. gingivalis induces NETosis in a gingipain-dependent manner. 38 Prevotella intermedia, part of the sub-gingival microbiota, is an anaerobic Gram-negative bacterium. P. intermedia plays a role in the initiation and progression of periodontitis by inducing a variety of pro-inflammatory cytokines such as IL-8, IL1β, proteases, and matrix metalloproteinases (MMPs), and macrophage inflammatory proteins. 39 P. intermedia does not express the PAD gene but facilitates human PAD activity via the induction of activated neutrophils as the main source of citrullinated antigens. 40 Two genes from P. intermedia have been characterized, nucA and nucD, encoding secreted nucleases. 41 Nucleases can block phagocytosis by neutrophils, which could lead to increased periodontal pathogenicity and the release of endogenous PAD. 40 In addition, this bacterium is known to induce the production of MMP-1 and MMP-8 in human periodontal ligament cells. These MMPs are also notably present in RA individuals. 42,43 P. intermedia increases the synthesis of prostaglandin E2 from arachidonic acid via inducing cyclooxygenase-2 (COX-2). In particular, overexpression of the COX-2 enzyme was detected in RA. 44 Fusobacterium nucleatum is a periodontal anaerobic bacterium associated with various forms of human diseases such as RA. The innate immune cells are made upby cells such as macrophages, neutrophils, mast cells, and NK cells contribute to the development of inflammatory responses in joints. 27 The binding of NK cells to F. nucleatum activates a wide range of inflammatory mediators implicated in the pathogenesis of the periodontal disease. 45 The balance between pro-/anti-inflammatory cytokines expression is maintained during periodontal full health. F. nucleatum exacerbates inflammation after the release outside the oral cavity or during dysbiosis.
For example, F. nucleatum induces inflammatory responses through the TLR4 pathway. 45 TLRs activation is very critical in the onset and progression of autoimmune diseases, such as RA. Patients with RA have exhibited raised levels of TLR2 and TLR4 in leukocytes. 46 Moreover, F. nucleatum has a powerful stimulatory effect on inflammatory cytokines, IL-6, IL-8, and TNFα. 45 APCs population, such as macrophages, and effector cells, increase inflammation and contribute to bone and cartilage destruction by releasing a variety of pro-inflammatory factors. 25 In particular, TNFα found crucial in the pathogenesis of the disease by increasing inflammatory cytokine levels, activating macrophages and lymphocytes. 47 Porphyromonas endodontalis is an anaerobic, gram-negative, black-pigmented, asaccharolytic bacteria associated with periodontitis and infected root canals. P. endodontalis has a lower ability to bind gingival cells than P. gingivalis because it does not harbor the PAD gene. 6,48 P. endodontalis also does not possess a trypsin-like enzyme associated with periodontal tissue destruction and does not display hemagglutination activity. 49 However, P. endodontalis produces collagenases and protease enzymes that can also play a key role during tissue destruction. 48 Another major virulence factor of P. endodontalis is LPS, which releases the secretion of inflammatory cytokines network and promotes bone destruction. 50 P. endodontalis was dramatically increased in the saliva microbiota profiles of patients with early-onset RA. 6 Tannerella forsythia is an obligate anaerobe related to periodontal disease. 51 59 Furthermore, LtxA induces the production of IL-1β and IL-18 cytokine by macrophages. These cytokines are the principal mediators in the immunopathogenesis of RA. 60 As well, LtxA promotes NET release via a CD11/CD18 (LFA-1) route. LFA-1, a cell surface receptor for LtxA on neutrophils, is involved in a downstream signaling cascade that may also participate in ROS generation. The increased production of ROSs is necessary for NET release. 61 The increased ROS following NETosis can activate the receptor activator of the nuclear factor kappa-B ligand (RANKL) expression on osteoblast and stimulate osteoclast activation. 62 The role of periodontal bacteria in ACPAs generation and RA development is depicted in Figure 1A, B.
Peptostreptococcus micros is an anaerobic Gram-positive bacteria predominated in periodontitis and infected dental root canals. 63 Yoshioka, et al. report that LPS released from A. actinomycetemcomitans can attach to P. micros, resulting in a population of bacteria that could be a potent stimulant for human macrophages. P. micros might help cytokine induction in a periodontal lesion, thus favoring inflammation. 64 P. micros can adhere to host epithelial cells and interact with periodontopathogenic bacteria, including F. nucleatum and P. gingivalis. 65 P. micros cell wall induced the secretion of a high level of IL-6 cytokine by macrophages. IL-6 enhances bone resorption. 63 Treponema denticola, an anaerobic oral spirochete, could be the main contributor to periodontal disease. 66 T. denticola Eno is a virulence factor, with higher degrees of homology with the human Eno. It is also served as a plasminogen-binding receptor, which assists in inflammatory cell invasion. 67  of pro-inflammatory factors and proteases. 72 F. alocis can convert the arginine to ornithine via an enzymatic pathway without the citrulline production step. 73 The L-ornithine-converting enzyme was increased approximately 1.5-fold in early-onset RA patients' microbiota, supporting a potential association between F. alocis and RA progression. 6 The link between oral dysbiosis with the pathogenesis of RA is depicted in Figure 2.

| THER APEUTI C INTERVENTI ON S
Although drug molecules, predominantly glucocorticoids, and DMARDs, are unchanged as first-line drug treatment; novel approaches are appealing options to address this challenge. 74 The most common type of adverse effect of DMARDs is an elevated risk of bacterial, fungal, and viral infections. 75 Figure 3A). 83 The use of complementary therapies such as PDT could improve the sense of well-being and increase the likelihood of remission. 77 PDT involves a photosensitizing agent, which is excited by visible light at specific wavelengths in the presence of oxygen. Next, the F I G U R E 2 Schematic illustration of the links between rheumatoid arthritis and periodontal Infections. (A) An oral dysbiosis onto the tooth surface initiates innate immunity by producing pro-inflammatory mediators in response to bacterial LPS (via the toll-like receptor). (B) Immune cells will produce pro-inflammatory mediators such as ILs, TNF, and MMPs, which also contribute to the augmentation of the immune response. IL-17 induces the production of pro-inflammatory cytokines and MMPs. The interactions among B and T cells and APCs play a major role in immune response activation, leading to the production of autoantibodies and the development of bone resorption. The autoantibodies contributed to the inflammatory process and resulted in bone and cartilage damage. LPS, lipopolysaccharide; ILs, interleukins; TNF, tumor necrosis factor; MMPs, matrix metalloproteinases; APCs, antigen-presenting cells.
excitation energy gives rise to ROS. 84 Therefore, PDT based on the photosensitizing agents induces cell death in inflammatory cells participating in the joint. Hence, combining PDT with standard treatments can improve the control of bone and cartilage damage in the treated joint. 77 Numerous studies reported that PDT significantly reduced pro-inflammatory cytokine levels such as IL-6, IL-17, and TNFα. 13,85,86 Nanotechnology can be a promising therapeutic strategy for RA.  Figure 3B). 90 Macrophages are dynamic cells that play a pivotal role during the pathogenesis of RA. The activated RA synovial macrophages express the folate receptor β (FRβ). Triptolide (TP) is a molecule with anti-inflammatory features, but its clinical application to treat RA has been limited due to many disadvantages like poor solubility, low bioavailability, and extremely high toxicity. Liposomes (Lips) are biocompatible spherical vesicles which used as preferred carriers for the delivery of therapeutic agents. 91 The main benefits of Lips include biocompatibility, high stability, high drug loaded to carrier ratios, nontoxicity, and protective features of drugs. 92  Lip. 94,95 The ASU combination inhibits nuclear factor kappa B (NF-kB), monocyte migration as well as decreases the side effects due to the co-delivery of ASU within Lips vesicles. 96,97 The loaded Lip with ASU has shown positive effects in controlling and reducing joint inflammation in osteoarthritis models. ASU/liposome combination may be effective in controlling RA joint degradations. However, further investigation is needed on RA models.
In addition, NPs can be used to overcome drawbacks and improve the potency and effectiveness of therapeutic agents. 98,99 The copper sulfides (Cu 7.2 S 4 ) NPs plus near-infrared (NIR) laser irradiation (808 nm, 1 W/cm 2 ) have been applied in PDT against RA.
While Cu-based nanomaterials can serve as photosensitizer agents.
The combination of Cu 7.2 S 4 NP with NIR light showed an increase in ROS production. Meanwhile, this combination reduces the level of pro-inflammatory proteins. 100 Zhao et al. showed PDT using tetra F I G U R E 3 Schematic illustration of the role of (A) Lactobacillus spp. and (B) nanostructures in rheumatoid arthritis therapy. Photodynamic therapy/siRNA complex nanostructures prevent the expression of pro-inflammatory cytokines and inhibit bone and cartilage destruction.
sulphonatophenyl porphyrin-titanium dioxide nanowhiskers have a strong therapeutic effect in RA. 13 Small interfering RNA (siRNA) showed great potential to decrease main inflammatory cytokines such as TNFα and IL-1β. 21 A siRNA does not easily deliver to cells through passive diffusion due to its high molecular weight, hydrophilicity, and negative charge. Of course, due to susceptibility to endonuclease cleavage, and short half-life in serum, an efficient delivery system is essential for the clinical application of siRNA. 101

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data are available from the corresponding author.

CO N S E NT FO R PU B LI C ATI O N
All of the authors approved the manuscript for publication.