“Mutualism” is a well-defined relationship that describes a form of cooperation between two living organisms of different species that ends up with a beneficial outcome for each one. Any disruption to such a relationship by an external trigger or a potential intruder puts at risk the well-being of both. In humans, oral and gut microbiota provide a noteworthy model of beneficial mutualism. Multiple recent evidences point to the possible pathologic consequences of a disruption to this ecosystem (altered microbiota profile or dysbiosis) on human well-being. The gut-joint axis found its clear way “Proof of Principle” in the pathogenesis of autoimmune rheumatic diseases including rheumatoid arthritis (RA), seronegative spondyloarthropathies, and Behcet’s disease in a number of studies. Current therapeutic trends are directed towards the diverse biologic and immune-pathogenic factors involved in the disease process. Addressing dysbiosis in RA features an attractive future therapeutic target. In this chapter, authors aim to explore the recent evidences regarding the pathogenic role of “gut dysbiosis” in rheumatoid arthritis (RA), highlighting the spectrum of immune-pathogenic events that might contribute to disease evolution and inspecting future directives of research.
- autoimmune rheumatic diseases
- rheumatoid arthritis
1. Rheumatoid Arthritis and The Microbiome Theory “The Revival of an Old Hypothesis”
Rheumatoid arthritis (RA) is a systemic immune-mediated chronic inflammatory disease that predominantly targets diarthrodial joints contributing to synovial inflammation, joint destruction, and deformity. The disease has been initially recognized as a complex genetically based inflammatory joint disease, a concept that was redefined toward a multifactorial immune-mediated etiology. The etiopathogenesis of rheumatoid arthritis is typically a challenge incorporating complex network of genetic factors (HLA genes), environmental and biological factors (infections, a dominant pro-inflammatory profile, dysbiosis), as well as individual habits (smoking) interacting to shape up disease phenotype [1, 2].
An infectious etiology of RA has been proposed for decades, Banntyne and Wohlmann in 1896 were the first to suggest a mycobacterial etiology behind the development of rheumatoid arthritis, a theory that awaited solid scientific evidence. The hypothesis of “molecular mimicry” and cross-reactivity between epitopes of microbial origin with a self-protein was among the proposed mechanisms to explain the role of infectious agents in the breakdown of immune tolerance in RA. Certain viral infections like Epstein-Barr virus (EBV), cytomegalovirus (CMV), and Parvovirus B19 have been additionally claimed as potential etiopathogenic agents in RA [3–9].
1.1. The oral sepsis hypothesis
In the 1900s, the oral sepsis hypothesis and its relation to arthritis led to the use of teeth extraction as a prevalent treatment of disease for several decades. The impact of polymicrobial phylotypes and the pro-inflammatory burden in periodontitis proved its contribution to the pro-inflammatory drive in RA. The
The infectious profile further extended to include other pathogenic bacterial species as wells as cell membrane proteins of mycoplasma in RA like
1.2. The infectious hypothesis and therapeutic evidences
The relationship between infections and arthritis was indirectly reinforced by the time antimicrobials showed an observable beneficial effect in the amelioration of arthritis. In the 1940s, sulfasalazine was the first rationally designed drug in the field of rheumatology, where RA was thought to be caused by Streptococci found in milk. Sulfasalazine was designed as a deliberate attempt to combine a sulfonamide antibiotic with a salicylate anti-inflammatory agent through an azo bond. Such “combination therapy” brought up good initial responses in RA as well as enteropathies. The triple DMARD therapy (a combination of methotrexate, sulfasalazine and hydroxychloroquine) currently stands among the first few recommendations in RA patients with poor prognostic features and moderate to high levels of disease activity [17–21].
Certain antibiotics like tetracycline have proven efficacy in the treatment of early seropositive RA which led to the approval of minocycline, as a DMARD [22, 23]. Clarithromycin is another example that gave observable beneficial effects in some refractory cases . Treatment with probiotics, particularly
2. The human gut ecosystem
Mutualism is a well-defined relationship between living organisms. The term describes a form of cooperation between two living organisms of different species that ends up with a beneficial outcome for each one. Partners in a mutualistic relationship make headway together with each forming a part of the other’s environment and maintaining its homeostasis in a typically harmless yet useful way. A disruption to such a relationship by an external trigger or a potential intruder puts at risk the well-being of both. In humans, oral and gut microbiota provide a noteworthy model of beneficial mutualism. Multiple recent evidences point to the possible pathologic consequences of a disruption to this ecosystem (altered microbiota profile or dysbiosis) on human well-being. The gut-joint axis found its clear way “Proof of Principle” in the pathogenesis of autoimmune rheumatic diseases including RA, seronegative spondyloarthropathies, and Behcet’s disease in a number of studies.
The term “microbiota” is defined as the ecological community of symbiotic, commensal, and pathogenic microorganisms that literally inhabit the human body and effectively contribute to human health and might contribute to disease. According to their contributions to human health, they can be classified to symbiomes and pathobiomes. The symbiome microbiota are groups of microorganisms predominantly bacterial as well as fungal (previously recognized as normal flora) that get acquired at birth and inhabit every part of the human body from the skin to mucosa (mouth, eyes, gut, and genital tracts). The alimentary tract represents an established microbial habitat for almost 29–30% of the overall commensal/symbiome population in the human body. They involve over 500 bacterial species encoding around three million different genes constituting a complex challenging ecosystem, difficult to differentially analyze or understand [28, 29].
2.1. The development of the human microbiota profile
The general composition of gut microbiota is much similar in most healthy individuals; however, the individuals’ microbiota profile retains a highly personalized pattern (fingerprint). This pattern helps to keep in situ homeostasis in healthy individuals; therefore triggers of dysbiosis are expected to offer a threat to the humans’ well-being in a variety of forms.
At birth: The initial shape up of the different microbial colonies of the gut begins as early as childbirth by the time the newborn gets exposed to microorganisms that inhabit the maternal genital tract during normal vaginal delivery (first encounter), maternal fecal bacteria, or the skin in the case of caesarian section. The fetal fingerprint of microbiota gets additionally shaped up by the skin as well as breast milk microbiota that add their signature to this microbial network .
Adulthood: The typical adult microbiota profile emerges by the end of the first year with more than 1000 different species from a dozen different divisions colonizing the gastrointestinal tract (4 × 1013 colonizing bacteria). The profile becomes increasingly stable with age. The acquired bacterial species pass through a series of modifications with colonies competing for survival, alternating their dominance aiming to establish phylogenetic richness and species diversity at the same time. Serial microbial evolutions are proven to be largely dependent on a multiplicity of host as well as environmental factors (the host genetics, the maternal microbiota, and mode of delivery as major determinants). Breast milk composition is another proven shaping factor of individuals’ microbiota profile (
2.2. The identification and characterization of gut microbiota
The human microbiome project served as a major contributor toward better understanding of the human microbiome. For a sufficient period of time, the conventional culture-based methods were the only methods available and routinely used for identification of gut microbiota, with over 500 bacterial species successfully isolated, cultured, and characterized from the human GI tract .
The majority of gut microbiota (over 80%) could not be technically cultivated ranking the culture-based methods as an investigative technique of limited value in determining the actual microbial diversity. The revolutionary era of the human microbiome project announced in 2007 and the utilization of novel culture-independent molecular techniques have successfully contributed to the in-depth understanding of such complex human microbiome network. Such culture-independent techniques target the highly conserved 16S ribosomal RNA (rRNA) gene sequence (a component the 30S subunit of prokaryotic ribosomes) of bacterial and archaeal microorganisms. Universal PCR primers targeting these conserved regions can be used for gene amplification to provide the complete nucleotide sequence of the 16S rRNA without prior knowledge of which bacterial species are present. Concomitantly, the 16S rRNA gene also contains hypervariable regions that provide species-specific signature sequences and enable unbiased bacterial identification utilizing next-generation sequencing platforms. Examples of these molecular techniques include quantitative polymerase chain reaction (qPCR), temperature or denaturing gradient gel electrophoresis (TGGE or DGGE), terminal-restriction fragment length polymorphism (T-RFLP), and fluorescent in situ hybridization (FISH). The next-generation sequencing and phylogenetic microarrays promote the in-depth analysis of the complete phylogenetic diversity of the intestinal microbiota, whereas the whole-genome shotgun sequencing is a technique currently applied for identification of the metabolic and enzymatic pathways present in these microbial communities. Such techniques were able to identify a portion of predominant species in the human gut [36–38].
Genomic culture-independent techniques disclosed that the human intestine is dominated by two divisions of bacteria, Bacteroidetes and Firmicutes, and both contribute to more than two-thirds of the total gut microbiota including anaerobes such as
2.3. The human-microbiota interdependence
Microbiota are involved in a number of structural, biological, protective, and metabolic processes including angiogenesis, postnatal intestinal maturation, and xenobiotic metabolism. They are additionally involved in the synthesis of vitamins, the digestion, and uptake of nutrients. Microbiota are capable of metabolically simplifying nutritional components through the process of fermentation; an important example is the indigestible carbohydrates (complex starch and dietary fibers) as well as short-chain fatty acids (SCFA, e.g., acetate, butyrate, and propionate) which constitute a critical prerequisite in a number of host physiological functions .
2.3.1. Microbiota and immunity
A unique harmonious and complex interplay exists between gut microbiota and the local as well as the systemic immune response in humans. Colonizing microbiota and their products have a profound effect on the development and maintenance of the immune system integrity [42–45].
22.214.171.124. The microbiota and T-cell differentiation, proliferation, and functions
Evidences support the existence of immune cross talks between the microbiota-derived products and the gut epithelium that involves molecular exchange of bacterial signals recognized by host receptors to mediate beneficial outcomes for both the microbiome and human beings [42–47]. The gut microbiota have the potential to effectively stimulate and direct the host innate and adaptive immune responses in humans by triggering instructional signals to the immune cells, particularly regulatory T (Treg)-cell and T-helper (Th) cell differentiation.
126.96.36.199. Short-chain fatty acids and Treg cells
SCFAs are actively produced by anaerobic microbiota in the colon as fermentation products of digestion-resistant dietary fibers [46, 47]. SCFAs produced locally in the colon are either absorbed and metabolized within the colonocytes or transported into the circulation to reach other organs, such as the liver and muscles. SCFAs exert direct and indirect regulatory effects on epithelial cells, antigen-presenting cells, and T cells via multiple mechanisms including metabolic regulation, histone deacetylase inhibition (HDAC inhibition), and G-protein–coupled receptor (GPCR) activation.
SCFAs can be converted to Acetyl-CoA and integrated into the citric acid cycle (Krebs cycle). Acetyl-CoA is a central molecule that stores energy and is eventually oxidized to CO2 for energy production (the cellular energy [ATP/ADP] level increases). This boosts a homeostatic T-cell sensor mammalian target of rapamycin (mTOR) to be activated in T cells which skews T-cell differentiation into effector T cells such as Th1 and Th17 cells at the expense of FoxP3+ T cells. mTOR activation also promotes the generation of IL-10+ cells. Thus, the SCFA regulation of cell metabolism and mTOR accounts for the increased generation of Th1 cells, Th17 cells, and IL-10+ cells.
The activation of SCFA-binding G-protein–coupled receptors (GPCRs) such as GPR41, GPR43, GPR109A, and Olfr78 initiates T-cell differentiation.
All major SCFAs such as C2, C3, C4, and C5 have HDAC inhibitor activity. Class I/II HDACs are major targets of SCFA inhibition in a concentration-dependent pattern. Treg generation was increased as a result of HDAC inhibition by SCFAs and histone H3 acetylation in key regulatory regions of the Foxp3 locus.
SCFAs can indirectly affect T cells through their effects on DCs. They suppress the development of bone marrow progenitors into myeloid DCs and inhibit functional maturation of DCs in vitro. For example, C4 suppressed the maturation of bone marrow-derived DCs and production of IL-12 but increased the expression of IL-23p19. Valproic acid, a branched short-chain fatty acid and potent HDAC inhibitor that suppressed the maturation of human DCs in vitro, inhibits the upregulation of T-cell–activating molecules such as MHC II, CD80, CD86, and IL-12.
These effects are combined to create the overall tolerogenic gut environment with a strong barrier function.
188.8.131.52. Microbiota and cytokine production
Microbiota produce stimulatory signals to gut macrophages to produce interleukin-1 (IL-1). IL-1 acts on type 3 innate lymphoid cells in the intestine, producing colony-stimulating factor 2 (CSF-2). CSF-2 induces myeloid cells (including dendritic cells and macrophages) to produce immune regulatory cytokines (retinoic acid and IL-10) which support the conversion and expansion of regulatory T cells (Treg) critical for maintaining immune tolerance in the gut.
Microbiota have the capacity to produce a certain set of microbial products—gut microbiota–derived butyrate and polysaccharide A PSA (e.g.,
The “segmented filamentous bacterium (SFB)” is another symbiotic gut commensal that is capable of inducing the appearance and activation of Th17 cells in the lamina propria, which secrete the pro-inflammatory cytokine (IL-17) and thereby enhance mucosal immune responses of the host .
184.108.40.206. Experimental evidences illustrated the impact of dysbiosis on the immune system and T-cell function
Multiple experimental evidences revealed that a germ-free environment is associated with a decline in neutrophil count and function [42–45]. Recognition of peptidoglycans from gut microbiota by the cytosolic recognition receptor nucleotide oligomerization domain 1 (NOD1) is capable of enhancing the killing activity of marrow-derived neutrophils contributing to immune integrity. A germ-free environment was associated with impaired neutrophil phagocytic function, superoxide function, and nitric oxide generation, which could not be restored upon pathogen exposure. Germ-free rats lack the microbe-derived ATP capable of stimulating dendritic cells (CD70 and CX3CR1) involved in the subsequent differentiation of Th17 cells. Additionally, in germ-free rats, systemic, not gut macrophages, were reduced, and gut macrophages lack macrophage-activation markers, such as MHC class II.
Early-life microbial exposures alter sex hormone levels and modify the progression to autoimmunity in the nonobese diabetic (NOD) mouse model of type 1 diabetes (T1D). Colonization by commensal microbes elevated serum testosterone and protected NOD males from T1D. Furthermore, transfer of gut microbiota from adult males to immature females altered the recipient’s microbiota, resulting in elevated testosterone and metabolomic changes, reduced islet inflammation and autoantibody production, and T1D protection .
3. The gut-microbiome checkpoints
Under normal conditions the human body has established a number of locally protective mechanisms and checkpoints to maintain immune integrity. These checkpoints include:
3.1. Mucosal buffer zone: “first-line defense”
Mucosal buffer zone represents an effective physicochemical barrier which is composed of the thick mucus layer, a number of antimicrobial proteins, and secretory IgA antibodies. The components of this barrier coalesce to minimize the contact and dilute any possible pathological microbial trigger between the commensal microbes in the gut lumen and intestinal epithelial cells that line the gut wall [50, 51].
3.2. Intestinal epithelial cells: “second-line defense”
Microbiota that escape the initial “buffer zone” encounter a second defense strategy represented by the tight junctions formed between the intestinal epithelial cells featuring a physical barrier.
Epithelial cells produce a variety of antimicrobial/bactericidal proteins, such as defensins, cathelicidins, and C-type lectins and express Toll-like receptors (TLRs) in their cell membrane. Toll-like receptors (TLRs) present on the surface of cells of innate immunity recognize pathogen-associated molecular patterns (PAMPs) with activation of the signaling adaptor molecule MyD88 (myeloid differentiation primary-response protein 88) provoking a downstream inflammatory responses. Lipopolysaccharides (LPS) are important components of the outer membrane of all Gram-negative bacteria. TLR4 is a natural ligand for LPS and upon binding to LPS leads to activation of cell and secretion of inflammatory cytokines. Cytokines in turn activate cells of the adaptive immune system that generates antigen-specific response .
3.3. The Innate and Adaptive Immune responses of the Lamina Propria “Third-Line Defense”
The composition and function of the gut microbiota play a critical role in regulating the Th17/Treg cell balance in the lamina propria. The innate cells in lamina propria constitute an important local line of immune defense that constantly surveys the gut lumen tracing undesirable antigens. The first is the intestinal macrophage system that phagocytoses microorganisms bypassing the first- and second-line barriers. The second is the lamina propria dendritic cells (DCs, antigen-presenting cells) that present foreign peptides complexed with MHC class II molecules for priming of antigen-specific B- or T-cell receptors to initiate an adaptive immune response.
T helper cells feature another key player when primed via a complex network of receptors, cytokines, and transcription factors enabling their differentiation into several pro-inflammatory or anti-inflammatory subsets. The type 1 T helper (Th1) response develops in response to intracellular pathogens, while type 2 T helper (Th2) and type 17 T helper (Th17) cells are predominantly stimulated after identification of extracellular organisms [53, 54].
4. Dysbiosis and autoimmune inflammatory arthritis
Multiple animal models of human autoimmune diseases (AD) suggest the direct involvement of commensal microbiota in disease development. A change to a single bacterial species and/or the entire community leads to an imbalance between the pathobiome and the symbiome immune responses with breakdown of self-immune tolerance provoking a number of ADs . Mono-colonization with
Finally, commensal bacteria can produce and secrete large amounts of adenosine 51-triphosphate (ATP) that can activate a unique subset of lamina propria cells, CD70highCD11clow cells. The CD70highCD11clow subset cells could express Th17-inducing molecules, such as IL-6, IL-23p19, and transforming-growth-factor-_-activating integrin AV, and induce Th17 differentiation .
5. Altered Gut Microbiota and Inflammation in Rheumatoid Arthritis.
The Th17 pathway is one of the main inflammatory pathways. Th17 cells and its signature cytokine IL-17 have been involved in mediating pannus growth, osteoclastogenesis, and synovial neo-angiogenesis. Circulating Th17 cells as well as the IL-17 are significantly elevated in patients with RA. IL-17 induces RANKL expression in human synovial fibroblasts, leading to the loss of the RANKL/OPG balance with subsequent enhancement of osteoclastogenesis and bone erosion. IL-17 also increases the production of vascular endothelial growth factor (VEGF) in rheumatoid fibroblast-like synoviocytes (FLS), contributing to the angiogenesis in rheumatoid synovium. Finally, IL-17 stimulates the expression of various pro-inflammatory cytokines (e.g., IL-1β, TNF-α, and IL-6) and matrix-degrading enzymes (e.g., matrix metalloproteinase (MMP)-1, matrix metalloproteinase-2, matrix metalloproteinase-9, and matrix metalloproteinase-13) in whole synovial tissue, synovial fibroblasts, and cartilage, thus promoting inflammation, extracellular matrix breakdown, and cartilage destruction during RA development [61–65].
The gut mucosa represents a strategic landscape promoting a variety of interactions between the environment and the human host. They possess the potential to trigger autoimmunity in diverse ways. When genetic and/or environmental factors alter the balance in the microbiota composition, dysbiosis ensues. The metagenomics shotgun sequencing technique has revealed significant alterations in the microbiota profile in patients with RA. Evidences illustrated that changes in the symbiotic relationship between intestinal microflora and opportunistic bacteria as well as bacterial translocation from the gut to nasal mucosa or urinary tract may additionally enhance the risk of developing RA as well as disease-related comorbid conditions. Partial depletion of natural gut flora by antibiotic in experimental animals aggravates CIA .
Potentially harmful microorganisms/pathobiomes (such as SFB or
5.1. Microbial colonies overpresented in RA
Lactobacillus fecal microbiome communities were found to be significantly increased in patients with early RA with those from healthy individuals  (
). The abundance of
|Vaahtovuo et al. ||Early RA (n= 51) vs. fibromyalgia (n= 50)||Stool||16S rRNA hybridization and DNA staining||Bifidobacteria --,
|Gul’neva et al. ||Treatment-naïve RA (n= 94) vs. healthy (n= 97)||Stool, dental, saliva||Metagenomic shotgun sequencing||
|Liu et al. ||Early RA (n= 15) vs. healthy (n= 15)||Stool||Quantitative real-time PCR||
|Scher et al. ||New-onset RA (n= 44) vs. healthy (n= 28)||Stool||16S rRNA gene and WGS sequencing||Prevotella copri ++, Bacteroidetes --|
|Zhang et al. ||RA (n= 30) vs. healthy (n= 30)||Stool||16S rRNA gene and WGS sequencing||Enterococci ++, Clostridia ++, Colibacteria ++, Lactobacteria --|
Prevotella copri species
A strong positive correlation was illustrated between the
5.1.3. Other forms of gut microbiota
RA patients showed a dysbiotic gut microbiota characterized by a decrease in
On the other hand, RA patients have significantly less bifidobacteria and microbiome of the
5.1.4. Protective microbiota species:
Prevotella histicola “ P. histicola”
Oral feeding of
6. Future perspectives
Multiple lines of evidence support the potential pathogenic role of gut dysbiosis in RA which makes gut microbiota a possibly promising new territory for drug targeting . Restoring balance of gut microbiota might contribute to the improvement of disease symptoms in RA. Experimental models demonstrated the ability of certain microbial colonies to drive inflammation such as
Thus, it is plausible to hypothesize that targeting the postulated pathobiomes might contribute to clinical improvement in RA. Despite the successful use of antimicrobials in RA, they lack microbial specificity in most studies.
“Mapping of the individual’s microbiota profile and tracking their metabolic signatures might serve in the future design of a predictive profile of the potential to develop inflammatory arthritis, anticipate disease course, severity and specify more therapeutic strategies.”
“Restoring balance in dysbiosis represents an advent toward novel treat to target therapeutic strategies in RA, a promising step toward an effective personalized medical approach.”
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