Trojans in Oral Environments: Evidence of Molecular Mimicry in Oral Immunity

Oral microbiome possesses more than 1000 microbial species that co-exist with human oral cavity. However, when there is an imbalance in microbial ecosystem, infection and inflammation occurs. Chronic inflammation produces constant antigen-cell presentation and reactivity T and B cell results in an adaptive immune response with high specificity cell-cell and antibody response producing an autoimmune disease by molecular mimicry. In this chapter, using just BLAST, shows self-epitopes (autoantigens) from different autoimmune diseases such as Systemic lupus erythematosus, Sjögren’s syndrome, neuromyelitis optica, Stiff-Person syndrome, autoimmune diabetes, autoimmune thyroiditis, myasthenia gravis, autoimmune gastritis, autoimmune hepatitis, myositis and rheumatoid arthritis that possess similarities with microbial epitopes belonging to oral microbiome acting has a computer trojan occult in a software package.


Introduction
Inflammation is a physiological response to any aseptic or septic injury to provoke the activation of immune response to enhance the healing [1]. This event began firstly by the recognition of pathogens-associated molecular patterns (PAMPs) [2], microbiota-associated molecular patterns (MAMPS) [3] or damage-associated molecular patterns (DAMPs) [4] by macrophages [1], mainly, leading the stimulation of innate immune response and the generation of acquired immune response producing a cellular and humoral immunity [5]. In this way, inflammation recognized pathogens via toll-like receptors (TLRs) to stimulate an immune response to remove these pathogens from the body [1] and acquired immunity memory by the antigen presentation mechanisms [6]. However, chronic inflammation can last for weeks, months, even years, provoking cycles of injury and healing causing irreversible tissue damage, being a risk factor for the development of autoimmune disease [7,8].
Oral microbiome contains innumerable epitopes similar to self-epitopes than cause crossreactivity immune response provoking the kill of microbe and self-tissue injury generating an autoimmune disease [9][10][11]. This phenomenon is known as molecular mimicry [12] or epitope mimicry [13].

Molecular mimicry
Molecular mimicry, term proposed firstly by Damian, is the theoretical probability that exist similarities in the molecular structures (amino acid sequence or conformational structure) between pathogens and the host producing a cross-reactivity immune response turn a defensive immune response into autoimmunity [8,[12][13][14][15][16].
However, molecular mimicry has been demonstrated as a common mechanism by microbes to elude immune response and may modulate biosynthetic or metabolic pathway of the host involved in the regulation of apoptosis, cell proliferation, inflammation and immune response [14,17]. Pathogens imitate host proteins and their interactions interfering with the cell functions at four different levels [18]: • Full length protein or domain.
• Structure with apparently sequence similarity.
The Toll/Interleukin-1 receptor (TIR) domain is an example of full length protein mimicry. When pathogens stimulate the TIR domain signalosome, a molecular pathway is activated to reach the NF-kB to produce inflammatory cytokines to modulate an immune response. In this manner, pathogens can interfere or inhibit this downstream pathway by the production of similar structures producing a negative regulation of TIR pathway, evading the host immune system neutralizing the TLR signaling for survival and proliferation [18].
In other way, structures with apparently sequence similarity can be interfered with the immune regulation, inflammation and wound healing [19]. In this manner, viral chemokine of Kaposi's sarcoma-associated herpesvirus is very similar to human chemokine CX3CL1 [20] causing the activation or inhibition of immune modulation in the host [21].
Pathogens have homologs of short amino acid sequences known as motif mimicry [22,23] composed of 3-10 residues with the capability to altered immune molecular pathways of the host [18]. One example of this mimicry is the bacterial guanine nucleotide exchange factors (GEFs), as Map and EspM2 of E. coli than can activated GTPases in the host [24,25], who regulates many cell function as proliferation, survival, differentiation, migration and apoptosis [18].
Interface mimicry is produced by short linear motif than may adopt altered conformations altering the global protein conformation, generating the pathogen evasion [18]. Human GTPases and Map of E. coli and SopE of Salmonella, can serve as an example of interface mimicry.

The molecular mimicry mechanism
During T cell development, naïve cells moved from the bone marrow to the thymus. In this organ occurs the positive selection, when T cell CD4 + CD8+ recognized the MHC on cortical thymic epithelial cells, they receive signals than let a CD4-and CD8-differentiation according to their affinity to MHC class I or II [26]. The process of thymic selection eliminates 99% of precursor cells by apoptosis, leaving 1% to reach the periphery [27].
In this case, if an external peptide (such as microbe) present similarity with the host peptides, activate T cells can be presented by dendritic or macrophages cells. And if the host peptide possesses similar structure, the T cell becoming autoreactive with self-antigen [27], could originate an autoimmune disease ( Figure 1).
The importance of interaction of peptide-MHC-TCR cannot be underestimated, because, antigen presentation plays an important role for autoimmune disease. The MHC class I binding area is closed, limiting the length of the presented peptides to 8-10 amino acids [28], however, MHC class II binding site is open and led peptides with 14-18 aa in length [28], but under certain conditions shorter peptides can be presented [29].

Autoimmunity
Autoimmunity is defined as a condition of loss of immune tolerance to self-antigens causing an autoreactive immune T and B cells that attack own organs provoking an aseptic inflammation and comprised more than 80 chronic diseases characterized by inflammatory reactions that can either be systemic or organ specific [30] and no cure exist for the majority autoimmune diseases and the treatment is based by control disease symptoms [31].
The early event in autoimmunity is the presentation of self-antigen derived peptides in complex with MHC class II to self-reactive T cells in an inflammatory environment where antigenpresenting cell, dendritic cell mainly, is activated and drives co-stimulation and development of pathogenic autoreactive T cell and autoantibodies, playing a critical role in breaking tolerance to self during an autoimmune disease, leading tissue and organ damage [31,32], produced by susceptible and aberrant genes, environment exposure, and failed immune regulation [30].
Dendritic cells are the responsible for the initiation of primary T cell responses imprinting the phenotype Th1, Th2, Th17, Treg population in response to environmental signals mediating the breach of T cell tolerance in many autoimmune conditions [31] involved in the activation of other autoreactive B cells [33]. Indeed, T cell help for antigens and can lead the activation of B cells that recognized the foreign antigen but also cross-react with self-antigen [34] producing and autoimmune disease.
T cells, for example, are important for the pathogenesis of rheumatoid arthritis (RA), particularly in the initial phase of autoimmune response, inducing the joint inflammation of the joints [3]. The Th17 cells are very important because they promote the development of autoimmune diseases by producing IL-17 promoted osteoclastogenesis in RA by upregulating RANK-RANKL expression on osteoblast, macrophages and synovial fibroblast [3,35] (Figure 2).

Autoantigens
Autoantigens can be defined as antigens that can be assumed to be targeted in an autoimmune disease [28] by the production autoantibodies by autoreactives B cells. Indeed, autoantibodyproducing B cell originated from T cell responses to foreign antigens thought molecular mimicry between microbial antigens and self-antigens [33]. Pathogens are recognized via TLRs by APC and they are phagocytosed in phagosome, digested and many microbial epitopes are exposed. MHC-II is mounted and microbial epitope is coupled in the MHC-II. In this case, epitope with similar characteristics with self-epitope (in violet) is mounted in MHC-II. The epitope is presented to naïve T or B cell, and a specific immune response is initiated. This immune response provokes the production of antibodies and cell-cell response by liberation of proinflammatory cytokines to the antigen presented. In this case, this immune response is addressed to pathogens and host tissue, with similar epitope, originating an autoimmune disease.
The literature describes many autoantigens for each autoimmune disease. Type 1 diabetes mellitus (T1DM) is a metabolic disease that is explained as an autoimmune disease in which the B-cells in the Langerhans islands of pancreas are destroyed by autoreactive T and B cells resulting in a null production of insulin [28]. Zinc transporter 8 protein, pancreatic and duodenal homeobox 1, chromogranin A, islet amyloid polypeptide are new discovered autoantigens that explain the pathogenesis of T1DM [36].
Rheumatoid arthritis (RA) is a chronic inflammatory disease with a strong autoimmune component that affect bones and joints with the concomitant destruction, associated with adverse morbidity, mortality, and socioeconomic consequences [39]. Autoantibodies such as rheumatoid factors (RFs) and anti-citrullinated protein antibodies (ACPAs) founded in serum samples obtained years before the onset of clinical disease [40,41]. Autoantigens may cause a self-reactivity of T and B cells by dysregulation of homeostasis of immune response acting as a trojan horses harming own body producing an autoimmune disease.

Searching trojans in oral microbiome
Microbiome is defined by Lederberg as "the ecological community of commensal, symbiotic, and pathogenic microorganisms that literally share our body space and have been all but ignored as determinants of health and disease" [42]. In silico tools have provided a powerful means of understanding the contribution of the human microbiome to health and disease opening a great field for oral immunologist. In the era of computer trojan horse, microbial epitopes with high similarities (in sequence and structure) with the host, can act as little sequences for the evasion of host immune system, even more, this trojans may cause a T and B reactive cells provoking an immune response for the microbial elimination and the origin of an autoimmune disease [43].

Trojans against connective
Systemic lupus erythematosus (SLE) is an prototype of autoimmune disease, affecting the connective tissue [44], with a great spectrum of clinical symptoms such as joints, kidneys, skin, to other manifestation, in fact, SLE is a nonpreventable disease and may be life-threatening [45,46]. Many autoantigens have been described to induce cross-reactivity immune response to SLE such as Ro52, Ro60, La, RNP-A, Sm-D3, and RNP-70 K. RNA-A and RNP-70 K, however, oral microbiome contain epitopes with similarities against SLE autoantingens ( Table 1).
Ro ribonucleoprotein 60 KDa (Ro60) is an autoantigen most prevalent in systemic autoimmune diseases as SLE and Sjögren's syndrome, and exist in unabundant ribonucleoprotein complexes stabilizing small RNA to prevent degradation [47,48]. This protein has a 6 aminoacids (aa) similarity against VWA domain-containing protein of Prevotella denticola ( Table 1). Small nuclear ribonucleoprotein 70 kDa, another autoantigen in SLE, is a small protein conforming the spliceosome complex. This protein has a 7 aa similarity against Bacillus cereus ( Table 1).

Trojans against nerves
Aquaporin 4 is an integral membrane protein that conducts water through cell membrane founded in nervous system. It is presented as autoantingen in neuromyelitis optica, an autoimmune disease consisting of a chronic inflammation and demyelination of optical nerve and spinal cord. This protein has similarities against glycerol uptake facilitator protein 2 of Streptococcus pneumoniae, MIP family channel protein of Prevotella oralis and MIP family channel protein of Enterococcus faecalis ( Table 2). Glycerol uptake facilitator protein 2 is a putative nonselective transport channel in the inner membrane of bacterium [49] and MIP family channel is a transmembrane protein transporting small molecules [50]. Both proteins are in external side of microbial cell membrane been more efficient form antibody-epitope complex.
Glutamate decarboxylase 2 is an autoantigen related in Stiff-Person syndrome, an autoimmune disease that affects nervous system. Glutamate decarboxylase of Enterococcus spp. possess 7 aa similarities against autoantigen ( Table 2).

Trojans against diabetes
Type 1 diabetes is an autoimmune disease in which the B-cells in the Langerhans islands of pancreas are destroyed by T and B reactive cells lacking the insulin production [28], affecting children and latent autoimmune disease of adults [51]. One of characteristics of this disease is the recognition of beta cell proteins as autoantigens such as preproinsulin GAD65, islet antigen 2 (IA-2), ZnT8, nonspecific islet cell autoantigens (ICAs), imogen 38, pancreatic duodenal homeobox factor 1, chromogranin A, islet specific glucose-6-phosphatase catalytic subunitrelated protein, heat shock protein 60 and islet cell antigen 69. IA-2, possess 6 aa with similar  Table 3) that is present to bind noncovalently to peptidoglycan and chitin in cell wall [52].

Trojans against thyroiditis
Thyroiditis is an autoimmune disease that destroys thyroid cells by reactive T and B cells. This disease is also known as chronic autoimmune thyroiditis and chronic lymphocytic thyroiditis. The pathology of thyroiditis involves the formation of antithyroid antibodies that attack thyroid tissue, causing progressive fibrosis [53]. One common autoantigen of many described in the literature is the thyroid autoantigen 70 k also known as Ku autoantigen [54]. Ku is an abundant protein in the body with multiple functions as replication, transcription and cell signaling [54]. Pilin isopeptide linkage domain protein of E. faecalis, ompA family protein of Prevotella sp. and aldo/keto reductase of T. denticola have small epitopes with high similarities with Ku autoantigen 70 k ( Table 4).

Trojans against myasthenia gravis
Myasthenia gravis is an autoimmune disease that attacks neuromuscular junction where synapsis occurs between nerves and muscles causing muscle weakness in patients [55]. Autoantibodies such as muscle-specific tyrosine kinase (MUSK), acetylcholine, agrin and lowdensity lipoprotein receptor-related protein 4 (LPR4) have been described in the literature [56,57]. MUSK is a transmembrane protein that contains three IgG domains and one cysteine-rich domain in the extracellular region and a kinase domain in the intracellular region [56] Table 5). This lactobacillus protein is present in cell wall in gram positives and negatives associated to penicillin-binding proteins [58].

Trojans against chronic autoimmune gastritis
Autoimmune gastritis represents approximately 5% of the whole spectrum of chronic gastritis and must be differentiated from the one associated with chronic Helicobacter pylori infection [59]. Gastritis is a chronic inflammatory disease involving gastric body and fundus, with the progressive reduction and/or disappearance of gastric glands that are sometimes replaced by intestinal or pyloric epithelium [60]. Autoantigens for the autoimmune gastritis has been related as Gastric ATPase α subunit, Gastric ATPase β subunit and Gastric intrinsic factor [61]. Gastric ATPase α subunit have three epitopes in different position in the same protein with a 6 aa, 7aa and 15 aa similarity, to Ca2 + Àtransporting ATPase of Streptococcus pneumoniae ( Table 6).

Trojans against liver
Autoimmune hepatitis is a chronic and progressive inflammation of the liver from an unknown cause, whose pathology is explained by the failure of immune tolerance in a genetically susceptible individual leading to a reactive T-cell mediated inflammation caused by various environmental triggers including infections, medications, and toxins [62]. Autoantigens for autoimmune hepatitis have been related such as O-phosphoseryl-tRNA(Sec) selenium transferase (SLA), cytochrome P450 2D6 isoform 1 (CYP2D6) and formimidoyltransferasecyclodeaminase isoform C (FTCD) [61]. FTCD epitopes have similarities with glutamate    Table 7).
Primary biliary cirrhosis (PBC) is now known as primary biliary cholangitis [63]. It is an autoimmune disorder which leads to gradual destruction of intrahepatic bile ducts resulting into periportal inflammation, cholestasis [63]. This disease is common among women of middle age worldwide. Primary biliary cirrhosis is associated with highly specific autoantibody [64]. The anti-mitochondrial antibody is found in 85% of the cases, other antibodies associated with disease is an antinuclear antibody (ANA), anti-multiple nuclear dot antibody (anti-MND), anticentromere antibody, pyruvate dehydrogenase complex E2 (PDC-E2) and antinuclear envelop antibody [61,63] . PDC-E2, possess 7 aa with similarities to dihydrolipoyllysine-residue acetyltransferase of Enterococcus spp.

Trojans against muscle
Myositis is an autoimmune disease that attack muscles [65]. There are three types of this disease: polymyositis, dermatomyositis, and juvenile myositis and possess and autoimmune origin, meaning the immune system is attacking the muscle [66]. This disease is not present in etiology. Although myositis is often treatable, these diseases are poorly understood and do not always completely respond to current medications [66]. Autoantigens has been related in the literature: histidine-tRNA ligase, cytoplasmic isoform 2, threonine-tRNA ligase, cytoplasmic isoform 1, exosome complex component RRP45 isoform 1, exosome component 10 isoform 1, chromodomain-helicase-DNA-binding protein 4 isoform 1, interferon-induced helicase C domain-containing protein 1, MORC family CW-type zinc finger protein 3 isoform 2, signal recognition particle 54 kDa protein isoform 2, E3 ubiquitin-protein ligase TRIM33 isoform alpha and 3-hydroxy-3-methylglutaryl-Coenzyme A reductase isoform 1 [61]. Threonine-tRNA ligase, cytoplasmic isoform 1 autoantigen, possess many epitopes with high similarities with threonine-tRNA ligase of Aggregatibacter actinomycetemcomitans and threonine-tRNA ligase of Streptococcus spp. (Table 8).  A. actinomycetemcomitans, and Streptococcus spp., present epitopes with high similarities of self-autoantingen Threonine-tRNA ligase, cytoplasmic isoform 1.

Trojans against collagen
Collagen is the most tissue presented in the body; it is associated with the skin, kidney, nerves, blood vessels and muscles protecting them against compressive forces [67,68]. Rheumatoid arthritis (RA) is a progressive autoimmune disease that affects directly the collagen by the chronification of inflammation causing a tissue damage (specially cartilage and bone), functional impairment, severe disability and premature mortality [69,70]. Periodontitis is a chronic disease by microbial multispecies insult. Microbiome of periodontal disease (PD) could be showed some bacteria such P. gingivalis, P. intermedia, Tannerella forsythia. F. nucleatum and Aggregatibacter actinomycetemcomitans, with epitopes that provokes autoreactivity against collagen [71]. Anti-citrullinated protein is an important autoantigen present in patients with RA having antibodies anti-Pg [72]. Obando-Pereda et al. showed that an epitope of Prevotella sp. has high similarity with human collagen report a positive antigen-antibody complex in RA and PD patient's sera [8] ( Table 9).

Conclusion
The majority of autoimmune diseases possess an unknown etiology and can be explained from genetic factors to molecular mimicry. In silico, tools for biological purposes are important to determinate if external epitopes that possess similarities with epitopes from autoantigens. Epitopes for Systemic lupus erythematosus, Sjögren's syndrome, neuromyelitis optica, Stiff-Person syndrome, autoimmune diabetes, autoimmune thyroiditis, myasthenia gravis, autoimmune gastritis, autoimmune hepatitis, myositis and rheumatoid arthritis, possess microbial epitopes belong to oral microbiome with high similarities that can explain the possible etiology of autoimmune disease by molecular mimicry.