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Autoimmune diseases, Autoinflammatory diseases, Self-tolerance, Danger Signal theory, Major Histocompatibility Complex (MHC), Protein Tyrosine Phosphatase 22 (PTPN22), Cytotoxic T-lymphocyte Antigen (CTLA4) , Familial Mediterranean Fever (FMF), BehÒ«et's disease (BD).
A percentage of the world's population inherit a combination of genes that induce susceptibility to various autoimmune diseases when triggered by an external factor. Autoimmunity can result due to a single gene defect or an association of various mutated variants resulting in a complex disease. Many genes associated with autoimmune and autoinflammatory diseases have been identified by gene detection techniques such as genome-wide association (GWA) study. Most of the diseases studied have HLA-associations. Some genetic predispositions including PTPN22 and CTLA4 have been proven to overlap between autoimmune diseases even though the clinical manifestations vary. A clear line between autoimmune and autoinflammatory diseases can only be drawn for a few diseases but there are still some diseases like Behcet's disease that share features of autoimmune and autoinflammatory diseases hence it is classified as a Mixed Pattern Diseases. This field of research is still a mystery with many questions that are unanswered.
1. Introduction to Autoimmunity and Autoinflammation
What is autoimmunity (definition)
Autoimmunity or 'horror autotoxicus' as described by Paul Ehrlich in the early twentieth century is when the immune system attacks the self epitopes on tissues hence resulting in an autoimmune disease, which is chronic and has a characteristic tissue damage (1). An autoimmune response is similar to a normal immune response in terms of being activated by an antigen (autoantigen), which triggers the production of antibodies (autoantibodies) by plasma cells and production of effector cells (3). For a disease to be defined as autoimmune, the resulting tissue damage should be mediated by autoreactive lymphocytes and/or their soluble products, proinflammatory cytokines, and autoantibodies as an adaptive immune response to self-antigens (5).
Autoimmune diseases are highly important as they play a major role in morbidity and mortality around the Globe. In addition to this, autoimmune diseases increase the economic burden on the country. During development in the thymus, T cells are monitored for self-reactivity. Those with autoreactive T-cell receptors are eliminated but this mechanism is not infallible. Some T cells with the ability to recognise self antigens are never completely eliminated by the immune system as they escape into the periphery (2). The self reactive lymphocytes are still able to recognise foreign molecules hence they play a major part in the conduction of an immune response. Therefore, eliminating these self-antigens would result in weakening the function of the immune system. Current evidence suggests that the CD25+CD4+ T cells are self reactive but this property is essential in the commitment to a TR-cell lineage. This shows that self-reactivity can be advantageous as part of a committed cellular mechanism preventing autoimmunity (4).
1.1a Breaking of self-tolerance proposed to define autoimmunity
The Generic definition of autoimmunity that was first proposed is that it is a self-directed tissue inflammation caused by breaking of self tolerance by the responses of the dendritic cell, B and T cell, responses in primary and secondary lymphoid organs ultimately leading to the establishment of an immune response to self antigens. In the concluding clinical expression of the disease, the response by the adaptive immune system plays the predominant role. Presence of organ-specific autoantibodies is found years before the clinical symptoms of the disease are seen and may be the direct cause of lesions in some disorders (1,13).
Self-tolerance is an important concept that must be understood before going into the depth of the genetic associations with autoimmune diseases. Tolerance is the process that eliminates or neutralizes autoreactive cells (autoantigens) and a failure in this process will result in autoimmunity (13).It is known that every individual possesses tolerance to self-antigens but this mechanism can flaw resulting in autoimmunity. After the substantial amount of work done on animal models to understand the mechanism of self-tolerance, use of CD4+ T cells were found to be the most appropriate as they gave the best result to the understanding of this concept (5).
The two types of self-tolerance are; central tolerance and peripheral tolerance. Tolerance that is induced in lymphocytes that are developing in the thymus for T cells and bone marrow for B cells is known as central tolerance. This is the first mechanism that was put forward to explain how the immune system is able to distinguish between self and non-self molecules. An immature lymphocyte that is able to recognise an antigen will undergo apoptosis or inactivation as a result of the negative signal generated, which would normally have activated a mature lymphocyte (5,6).
Another mechanism that is proposed for the self/non-self discrimination is the activation of naive mature lymphocytes to antigen-receptor signals in the early stages of an infection caused by an increase in antigen concentration produced by a foreign invading pathogen. The naive lymphocytes have been tolerized to self proteins by strong signals as they are constantly exposed to self proteins in every cell of the body (6).
The third mechanism involves the innate immune system. Mature lymphocytes that are found in the peripheral tissues may be exposed to self antigens, which will lead to a negative signal that will inactivate or kill the self-reactive tissue. This is called peripheral tolerance and it occurs in the absence of antigen presenting cells hence no costimulatory molecules are produced. Principle mechanisms of peripheral tolerance include; deletion by apoptosis, functional unresponsiveness also known as anergy and suppression by regulatory T cells (5).
In summary, self-nonself discrimination involves both intact arms of the immune system. The innate immune system recognises Pattern Associated Molecular Paterns (PAMPs) by pattern recognition receptors (PRR'S) hence co-stimulatory molecules are triggered, and the adaptive immune system triggers these through recognition by rearranging receptors that recognise molecules expressed on target cells (11).
1.1b The four main checkpoints to prevent autoimmunity
However, there are many tolerance checkpoints that exist in the immune system to prevent self-antigens from stimulating the persistent growth of self-reactive B and T lymphocytes (8).This mechanism is described in greater detail below. These checkpoints work together to ensure that autoimmunity is prevented without hindering the action of the immune system to foreign pathogens. Autoimmunity will only arise if these checkpoints are conquered to cause the development of effector T cells that will target the tissues (5,6).
Firstly, any cell that displays a receptor to recognise self molecules is targeted to die. Secondly, any cell that has a self-reactive receptor has to undergo somatic hypermutation and another stage of V(D)J recombination to form a receptor that is not self-reactive. The third checkpoint involves clonal anergy, which is changing the intrinsic biochemical and gene-expression to prevent the self-reactive receptor from recognising a self cell. Finally, the extrinsically regulated mechanism; 'immunological ignorance' is the last checkpoint that stands in the way of production of autoantibodies and effector T cells to destroy self tissues (5,6). This is illustrated in figures 1 and 2 below.
Figure 1: This diagram is taken from Goodnow et al. (9) to show the checkpoints that exist at multiple stages in the evolution of the immune response. The descriptions of each of the numbers on the figure are shown above and are taken from the article.
Figure 2: The figure above has been taken from Goodnow et al. (7) to illustrate the four protective checkpoints described above.
Despite these protective mechanisms, some individuals end up with an escape of the self-reactive lymphocytes thereby the activation of autoreactive T cells will eventually lead to autoimmune-mediated tissue necrosis as mentioned earlier. This protective system is not infallible and factors such as; genetic and environmental factors, play a key role in the development of an autoimmune disease but this mechanism is yet to be understood (5,6). For further understanding, it is worth considering how the initial T-cell activation is controlled. Antigen-presenting cells (APC's) play a key role in activating Naive T cells by binding to antigens and sending signals in form of cytokines and costimulatory molecules such as; B7 and tumour necrosis factor (TNF) family members (6).
1.1c The Danger Signal theory proposed to define autoimmunity
The first definition of autoimmunity involving the self/non-self discrimination created a few dilemmas about the concept involved. When focussing on self-directed tissue inflammation, associations of diseases that are classified as autoimmune with Major Histocompatibility Complex (MHC) and presence of autoantibodies is not clarified. Another difficulty with the concept is that it does not justify the presence of autoreactive lymphocytes in most people that do not lead to an autoimmune disease. In addition to this, the concept does not account for tumours that possess foreign mutated epitopes not being rejected by the immune system. Finally, foetus' are not rejected either and this is not supported by the first theory proposed to define autoimmunity (1).
To counter for these problems, Matzinger (3) proposed an alternative theory called the 'danger signal theory', which in turn explains the function of the immune system in terms of danger signals such as exogenous pathogens and endogenous damaged tissues causing activation instead of self/non-self discrimination. However, the problem with this model is that it does not strictly outline the specificity of autoimmunity to the adaptive immune system (1,3). Taken from Matzinger (3); the danger model shifts control of autoimmunity to the tissues that are in need of protection rather than the cells that protect them. This model therefore eliminates the self/nonself discrimination proposed earlier and highlights the importance of danger signals (dangerous to the body) in triggering autoimmunity.
Brief overview on Genetics
1.2a Mendelian disease versus Complex disease
Mendelian diseases are defined as the diseases that are caused by a single gene opposed to Complex diseases, which result from interaction of multiple genes with the environment. Environmental factors can include; drugs, toxins, infections due to pathogen exposure. It is important to realise that up to date, only the rare autoimmune and autoinflammatory diseases have a strong genetic link with Mendelian diseases of inheritance (7). It is also important to note that most autoimmune diseases are complex in nature and not all the susceptible individuals will be affected (Incomplete Penetrance). Figure 3 below taken from Rioux et al. (5) is a model to show how single gene disorders are different from complex diseases in terms of autoimmunity. The explanation of the model is taken straight out of this paper.
Figure 3: a, In simple mendelian traits, the relationship between the causal genetic variant (genotype) and the disease state is deterministic. b, In complex traits, the clinically recognized disease state results from interactions between multiple genotypes and the environment. Individual genotypes can affect one or more components of the adaptive or innate immune systems; together these lead to an altered immune response to self antigens. On the basis of current findings, the influence of any individual causal allele is modest, and therefore the relationship between the causal variant and the disease state is probabilistic. Although still providing an incomplete picture, the genetic discoveries in mendelian and common diseases are beginning to help build a model of autoimmune disease. The ultimate goal is to build a specific model for each individual disease whereby the effect of individual risk factors (genetic and non-genetic), their interactions, and their impact on disease susceptibility, disease progression and clinical management, are understood (5).
1.2b Genetic Predispositions associated with autoimmune diseases
The association of genes with autoimmune diseases have been shown in animals such as inbred mouse strains that are prone to a certain disease for example type 1 diabetes. Mice that possess the NOD strain have a high chance of getting diabetes however; female mice stand a higher chance of becoming diabetic than male mice. Type 1 diabetes in known to run in families, which has been proven by monozygotic (identical) twins both becoming diabetic whereas this is not seen in dizygotic (nonidentical) twins (5).
It has been found that there are some genetic variants that predispose to multiple autoimmune diseases. This clearly indicates that these diseases have common pathways of pathogenesis despite the differences in their clinical manifestations. However, some autoimmune diseases do not share predisposition by the same genetic variants and this would be indicative of the existence of different pathways to pathogenesis. It has also been found that most autoimmune diseases have multiple genes involved in the predisposition to the diseases hence they are complex diseases. There is also heterogeneity among subphenotypes within a disease and across major racial groups (8). It still remains unclear to what extent common variation versus multiple rare variants contribute to disease susceptibility (8). However, some of the actual causative genetic variations that explain the associations have not been definitely established.
Table 1 attached below shows the genetic associations with various diseases that have been confirmed as they met statistical significance. Clues to the underlying mechanisms and pathogenesis have also been suggested for these diseases. This article will focus on PTPN22 and CTLA4 listed in the table. Until very recently, three basic methods have been used to identify genetic variants that may contribute to any human phenotype, including autoimmune diseases. GWA (Genome-Wide Association) scan approach was used to find most of these genetic associations that are listed in the table. Many of these genetic associations were also discovered using candidate gene approaches or genetic linkage analysis (8).
Candidate gene association studies are usually done to address a possible hypothesis, this means that a gene with a known function and a potential to influence disease phenotype is investigated. However, Genetic linkage analysis compares a marker and gene in unrelated cases and controls for non-random association. This method depends on the cosegregation of chromosomal regions with a phenotypic trait within families, as is typical for highly penetrant Mendelian disorders (8). In the past few years, GWA scans have been used the most as they have been the most effective method for genetic mapping for autoimmune diseases and it has resulted in brilliant genetic association discoveries. Unlike candidate gene approach, there is no association to a particular hypothesis in a GWA scan but both methods have a recognized association (8).
Table 1: This table shows genetic associations that have been confirmed with autoimmune diseases (focus of article will be on the genetic predispositions and the diseases associated with these shown by the arrows on the table). Abbreviations: AITD, autoimmune thyroid disease; AS, ankylosing spondylitis; CD, Crohn's disease; GD, Graves' disease; MS, multiple sclerosis; PSA, psoriatic arthritis; PSO; psoriasis; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus; T1D, type 1 diabetes.
1.2c The Major Histocompatibility Complex (MHC) and autoimmune diseases
The susceptibility to over 40 diseases has been influenced by the genes of the human leukocyte antigen (HLA) region, the major histocompatibility complex (MHC) of humans located on the short arm of Chromosome 6 (5). It is the most widely studied region in the human genome because it has variants that are associated with autoimmune, infectious, and autoinflammatory diseases (12). The mechanisms underlying MHC association in autoimmune disease are not well understood. It is suggested that these genes control a number of functions involved in an immune response as well. HLA gene products are important for antigen presentation to cells of the adaptive immune system and for controlling activation of natural killer (NK) cells as well as other functions of the innate immune system (5). Theoretical studies in the development of models to determine the modes of inheritance of the HLA-associated diseases have led to a better understanding of the inheritance patterns in insulin-dependent diabetes mellitus (IDDM), rheumatoid arthritis (RA), multiple sclerosis (MS), ankylosing spondylitis, hemochromatosis, celiac disease, and others but the role of HLA in the pathogenesis of theses diseases remains unclear. It is now clear that many of the HLA-associated diseases involve heterogeneity in their HLA components, as well as non-HLA genetic factors (32). Autoimmune diseases are associated with HLA class II alleles. Current information suggests that there is still a lot of research to be done to find the valuable genetic insights associated with MHC. Table 2 below shows various diseases and their HLA-associations.
Category of disease
HLA alleles associated
Rheumatoid Arthritis(RA): non-organ specific
Classic Polygenic Autoimmune
HLA-DRB1, -DR4 (12)
Type 1 Diabetes (T1D): organ specific
Classic Polygenic Autoimmune
HLA-DRB1, -DR3, -DR4, -DR9, -DQA1, -DQB1 (12,33)
Systemic Lupus Erythematosus (SLE): non-organ specific
Classic Polygenic Autoimmune
HLA-DR3, -DR2, -DRB1, -DQA1, -DQB1 (12)
Classic Polygenic Autoimmune
Multiple Sclerosis (MS): organ-specific
HLA-DR2, -DRB1*1501, -DR3, -DR4 (12)
Behcet's Disease (BD)
HLA-B51, HLA-B57, HLA-B27
Crohn's Disease (CD)
HLA-DRB1, -DR7, -B18, -B21, -DR6, -DR8, -DR10, -B27 (12)
Table 2: List of various diseases and the named HLA associations.
1.2d Autoimmune disease association with PTPN22 620W allele
The most relevant and commonly associated intracellular signalling molecule with autoimmune diseases is with PTPN22 gene (intracellular tyrosine phosphatase) is located on chromosome IPI3.3. It encodes a lymphoid-specific phosphatase (Lyp) that binds to an intracellular tyrosine kinase, Csk. Csk phosphorylates Lck resulting in an inhibition of Lck kinase activity. A mutation in the PTPN22 620W results in reduced binding of Lyp to Csk. The overall effect is an increased number of autoreactive T cells during thymic maturation or dysfunction of regulatory T cells associated with the downregulation of early T-cell receptor signalling (30).
The first association of PTPN22 was with autoimmune type 1 diabetes (T1D) using a candidate gene approach focussing on R620W amino acid polymorphism that had the predicted functions. This was reported by Bottini et. al (36). Later on, PTPN22 was selected for a genome wide screen of likely functional variants in several candidate genes, which led to the association with RA (37). For both of these diseases, the PTPN22 620W allele confers nearly a two-fold risk of disease hence it is the second important genetic link to these diseases after MHC (8).
Other diseases that have been found to be associated with PTPN22 allele are Myasthenia Gravis (38), Grave's disease (GD) (39), Hashimoto's thyroiditis (40), systemic sclerosis, Addison's disease and a weaker association with idiopathic arthritis and SLE (41). All these diseases have a prominent component of the humoral autoimmunity. It has been found that PTPN22 leads to elevation of thresholds for TCR signalling with the overall expression that reduced, rather than elevated, T cell triggering and this may be a part of the phenotypic predisposition to autoimmunity. Elevated thresholds for receptor triggering have also been reported for B cells (8).
In contrast to these findings, Multiple Sclerosis (MS) has not been proven to be associated with the PTPN22 620W allele and this allele is protective for Crohn's disease (CD). The exact mechanism for this gene association is still unresolved and there is a high possibility that there may be multiple mechanisms (8).
1.2e Autoimmune disease association with Cytotoxic T lymphocyte antigen 4
For most autoimmune diseases, the inheritance pattern is complicated. MHC has been implicated as the major genetic component in the predisposition to these diseases but other genes are involved (16). Genetic variations in costimulatory molecules are involved with autoimmune disease susceptibility. Based on function and experimental data, the gene encoding Cytotoxic T lymphocytes antigen 4 (CTLA4) that maps onto chromosome 2q33 has been suggested a candidate gene for conferring susceptibility to autoimmunity CTLA-4 is expressed on activated CD4+ and CD8+ T cells and it binds the same ligands, B7-1 and B702, as CD28 but with about 20- to 50- fold higher affinity. CTLA-4 functions to downregulate the function of T cells unlike CD28. CTLA-4:B27 plays a critical role in the regulation of self-tolerance hence in the susceptibility to autoimmune disease (16).
The Cytotoxic T lymphocyte antigen 4 (CTLA4) associations with T1D, autoimmune thyroid disease and other endocrinopathies have been confirmed by GWA scans. The mechanisms and alleles involved have not yet been proven but there is a biological therapy targeting CTLA4, and their efficacy provides further confirmation of how important this pathway is (8). CTLA-4 is a negative regulator of T-cell activation hence it plays has an important function in immunologic homeostasis. Not much has been found on the regulatory mechanism for CTLA-4 expression. This inhibitory receptor expressed by T cells and this inhibitory signal determines whether T cells become activated and also affects the clonal representation in an immune response (34). CTLA-4 is expressed by T cells that recognise CD80 and CD86. The ligation of these costimulatory molecules will switch off T-cell responses and promote anergy (5). CTLA-4 is highly expressed by T regulatory cells and could have an influence in their function (35).
1.3 Proposed Definition for Autoinflammation
Autoinflammation is also a self-directed tissue inflammation but it shows more predominance to the innate immune system. The local factors at disease-prone sites cause the activation of the innate immune cells including macrophages and neutrophils (1). These activated cells will then target the tissues to cause tissue destruction. For example, in periodic fevers, there is disturbance of homeostasis of canonical cytokine cascades (1). Based on the generic definitions of autoimmunity and autoinflammation, figure 4 below has been taken from Mc. Gonagle et al. (1) to show the different categories that various diseases fit in.
The autoinflammatory diseases have a characteristic of an unprovoked inflammation whereby high-titer autoantibodies or antigen-specific T cells do not play a major etiologic role (15). Most of the autoinflammatory diseases are monogenic in nature. Galon and his colleagues (15) suggested that the polygenic diseases that shared features seen in Hereditary Periodic Fevers such as Familial Mediterranean Fever (FMF), which will be discussed later on, and lacking autoantibodies and MHC associations could be termed autoinflammatory disease (1).
Figure 4: The figure above taken from Mc. Dermott et al shows classifications of various diseases in subgroups.
Difference between Autoimmunity and Autoinflammation based on their
Autoimmunity has boundaries that are set by mutations associated with the monogenic autoimmune diseases, which show an increased susceptibility towards adaptive immune responses and it is acknowledged by the presence of autoantibodies. However, autoinflammation is defined by the mutations in the cells or molecules involved in the innate immune response at disease-prone sites (2). The concept of autoinflammatory disease was used to describe a group of inherited disorders characterized by episodes of unprovoked inflammation that lacks high-titer autoantibodies or antigen-specific T cells like in autoimmune diseases (19). Table 3 below is also taken from Mc. Gonagle et al. (1) and it clearly outlines the key differences between autoimmunity and autoinflammation.
Table 3: The key differences between pure autoinflammatory diseases and pure autoimmune disease are shown in the table above.
2. Classic Polygenic Autoimmune disease : Multiple Sclerosis (MS)
Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system, with pathological features similar to an autoimmune disease. It leads to substantial disability through deficits of sensation and motor, autonomic, and neurocognitive function (22). These symptoms are diverse and include; tremor, nystagmus, paralysis, speech and vision disturbance. MS has been regarded as a disease with multifactorial etiology, which means the susceptibility is influenced by multiple genes and environmental factors such as viral infections as a trigger and therefore is complex disease (14,22).
It has been found that geographical location, ethnicity, and clustering in temperate climates, all have an indirect contribution to MS susceptibility. The North European populations appear to be more susceptible to MS than those from a more tropical environment as shown by statistical analysis. 1 in 1000 people of north European origin who reside in temperate climates are more likely to develop MS (14). The age of onset is between 20 and 40 years of age and it affects atleast 350,000 people in the United States alone hence it has a high influence on the economy of the country (14,22). The incidence of MS is highly prevalent in females compared to males (2:1 ratio). However, this bias in gender has not been explained by X inactivation (42).
The etiology still remains unclear but interactions of genes and environment lead to the development of MS. The strongest genetic risk component of MS is found in MHC; HLA class II region (HLA-DRB1*1501, DRB5*0101, DQA1*0101, DQB1*0602), probably by their role as antigen-presenting molecules to pathogenic CD4+ T cells but it only accounts for 50% of the genetic susceptibility hence there should be other susceptibility loci and environmental factors. GWA studies have provided supporting evidence for a significant effect of some of the non-MHC genes including; IL2RA, IL7R, EVI5, CD58, KIAA0350, and RPL5 genes (21,22,43).
MS was simply a CD4+ Th1 cell-mediated autoimmune disease before recognition of the complexity of the disease and involvement with environmental factors such as viral infections was known. The activation of CD4+ autoreactive T cells and their differentiation into a Th1 phenotype are crucial events in the initial steps, and these cells are probably also important players in the long-term evolution of the disease. The target tissue is the Central Nervous System (CNS). Damage of the CNS is mediated by antibodies, complement, CD8+ T cells, and factors produced by the innate immune cells (22). Immunologic destruction of myelin basic protein (MBP) throughout the nervous system is the major pathology of MS (14). Components of; the innate and adaptive immune system, CD4+ T cells and CD8+ T cells and antibodies all contribute to different aspects of the disease process (22).
Many therapeutic agents for MS have been tested but the disease is complex and unreliable to manage. Older agents include Methotrexate, Cytoxan, Imuran and recently IFM-β and novel strategies such as use of T-cell receptor peptide immunization.
3. Rare Monogenic Autoinflammatory diseases: Familial Mediterranean Flu
FMF is a systemic monogenic autoinflammatory disorder characterised by unprovoked one-to-three day (recurrent) episodes of fever with localized inflammation that usually involves peritoneum (peritonitis), skin (rash), joints (arthritis) or pleural inflammation without high-titre autoantibodies or antigen-specific T cells (9,16,19). Amyloidosis is common in FMF and was a frequent cause of death in FMF patients. It is the result of tissue deposition of misfolded fragments of serum amyloid A (SAA), one of the acute-phase reactants produced by the liver in response to inflammation (20). These attacks occur frequently (few weeks or months) hence it is the most frequent and thoroughly studies hereditary recurrent fever (HRF) syndrome (19). FMF and other monogenic autoinflammatory diseases all show disturbances in pathways associated with the innate immune cell function, encompassing abnormal signalling in key cytokine pathways that include TND and interleukin-1 (IL-1β) via adaptor molecules termed inflammasome, as well as through mutations in proteins associated with bacterial sensing (1).
FMF is more prevalent in the eastern Mediterranean multiple populations and Middle East populations including Arabs, Jews, Armenians and Turks. The highest predominance is seen in Turkey and Japan. However, recently FMF mutations have been found in Spanish, Italian, Greek, Portugese, non-Jewish Caucasian, Indian and Chinese populations (16). In most people the age onset of the disease is before twenty years.
The mediation of FMF inflammatory symptoms is by the massive influx of polymorphonuclear leucocytes into the tissues that are affected, neutrophils, and a rapid acute phase response involving elevation in acute phase reactants (C-reactive protein, erythrocyte sedimentation rate, white blood cell count), which are still elevated between the attacks. This shows that there is ongoing inflammation. Elevation of other mediators including; interleukin (IL)-6, tumour necrosis factor (TNF-α) is seen during the attacks (18). The MEFV gene is involved in inflammatory reactions through altered leukocyte apoptosis, secretion of IL-1β, and activation of nuclear factor-κB pathway (18,20).
3.4 Genetics and FMF
The gene that is responsible for familial Mediterranean Fever (FMF) is the MEFV gene, located on the short arm of chromosome 16 (16p13.3) and identified by positional cloning in 1997 (16,18). The pathogenesis of this gene has only recently been found. A missense mutation (single amino acid substitution) in this gene is the cause of FMF. It is an inherited Autosomal Recessive disorder and has been found to be linked with environmental factors (9). MEFV gene encodes a protein called pyrin or marenostrin and most of the mutations are found on the C-terminal end of the protein encoding B30.2 domain (16).
Pyrin is a major regulator of the inflammasome platform controlling caspase-1 activation and IL-β processing (17). This gene plays an important part in apoptosis, inflammation, and cytokine processing regulation. The N-terminal end of pyrin is the pyrin domian, which modulates the innate immune response. Through the homotypic domain interactions, pyrin binds to the common apoptosis-associated speck-like protein (ASC) and contributes to the activation of pro-caspase-1 resulting in the regulation of Il-1β, and IL-18 secretion. Most of the FMF-associated mutations are located on the B30.2 (SPRY) domain. This domain has a function in signal transduction and ligand binding; at the carboxy terminus of the protein hence a mutation in B30.2 may lead to postponed apoptosis and inflammation due to the reduced ability of pyrin to control IL-1β activation. A mutation in MEFV gene will lead to an abnormal formation of pyrin protein, which acts as a susceptibility factor in inflammatory conditions (18).
FMF and Behcet's disease have been associated. There have been increased frequencies of MEFV mutations reported in Behcet's patients, and, conversely, Behcet's disease has been reported at an increased frequency among Israeli patients with FMF (20).
Therapeutic goals include suppression of acute atatcks and preventing long-term amyloidosis. Anakinra is a recombinant human interleukin (IL)-1β receptor antagonist used as a first-line drug as IL-1β plays a role in the pathogenesis of FMF. Another treatment used is daily oral colchicine therapy, which has been effective in preventing acute attacks of FMF and development of amyloidosis. TNF inhibitors; etanercept, has been effective in reducing the clinical and laboratory evidence of inflammation (20).
4. Mixed Pattern Disease: Behcet's Disease (BD)
Behcet's disease (BD) is a multiorgan disease of an unknown etiology characterized by immune-mediated occlusive vasculitis. The major symptoms of BD involve ulcers and lesions, which include; recurrent oral aphthous and genital ulcers that lead to scarring eventuall, lesions in the eyes (uveitis) and skin lesions. Lesions of the eye and involvement of the brain and spinal cord can be said to be the most serious symptoms of this disease. Other manifestations involve vasculature, gastrointestinal
tract and the nervous system. These symptoms will vary in different people due to the differences in ethnicity, geographical and individual differences (27,28,29). It is an important disease because it contributes strongly to the morbidity and mortality as well as being an economic burden to the countries with a high prevalence of this (29).
BD has been found to be mostly prevalent in an area that coincides with the ancient Silk Route (29), which consists of countries in the Middle East, East Asian countries and the Mediterranean countries. In Turkey, the country with the highest prevalence, there will be approximately 370 patients per 100,000 of the population. The prevalence decreases up to 150 times in Asia and it decreases even further in Europe and Asia (27). Recently it has been found that BD is not rare around the world. The age of onset of BD has been found to be around twenty to thirty years but there have been cases in every age group. BD used to be more prevalent in males but in the last two decades this balance has changed to result in an equal prevalence in both of the sexes (28).
Presently, BD has an unknown aetiology. However, some evidence has been found on an excess response to pathogens and an increased cytokine and chemokine production. This may explain the vasculitis symptom associated with BD but how BD is triggered initially is still a question to be answered (29).
In the laboratory, serum samples from BD patients' show increased levels of cytokine. This certainly explains the hyperactivity of the immune response in these patients. The elevated cytokines found include; IL-1β, IL-1RA, IL-2, IL-4, IFN-γ, IL-6, IL-8, IL-10, IL-12, IL-13, IL-18, TNF-α, sTNFRII, IFNα, macrophage inhibiting protein-1α and GM-CSF. The mRNA analysis of the oral and genital ulcers showed elevated IL-12, IFN-γ, and MCP-1 in comparison to normal skin (28,29). The erythrocyte sedimentation rate and C-reactive protein (CRP) is usually moderately raised. Sometimes serum immunoglobulins are elevated. However, autoantibodies such as rheumatoid factor (RF), antinuclear antibody (ANA), anticardiolipin and antineutrophilic antibodies (ANCA) are absent (27).
Anterior uveitis and joint lesions in patients with BD comprise mainly of a neutrophilic infiltrate which, does not lead to tissue damage but is self-limiting, and driven by CXCL8. Synovial fluid from BD patients contains significantly lower levels of IL-1β and TNF compared with RA samples. The complex nature of BD may be explained by the persistent presumed Th-1 mediated mucosal lesions causing an elevated systemic production of CXCL8 and TNF, which induces a vasculitis.
CD28 is found in the serum of active BD patients therefore it may be a good marker for BD. CD28 is correlated with high C-reactive protein, which decreases with inactivity (28).
γδ T lymphocytes have a role in the immune response to infections and in auto-immunity by recognizing bacteria-derived and autologous antigens. Patients with BD have increased numbers of these T cells in circulation and mucosal lesions. γδ T lymphocytes have an activated phenotype in BD (they express activation markers, like CD 25, CD 29 and CD 69) and produce inflammatory cytokines, including IFN-γ, TNF-α and IL-8. Culture of γδ T lymphocytes from BD patients proliferates in response to mycobacterial HSP-derived peptides and in response to products from microorganisms in oral ulecers. Antigen presenting cells (APC) produce IL-12, so it is likely that they are involved in the Th 1-type polarization in BD. They also produce IL-18 which has been shown to increase neutrophil function. Neutrophils are hyperactive in BD, with increased chemotaxis phagocytosis, superoxide production and myeloperoxidase expression and produce several cytokines, including IL-12. The precise mechanism of neutrophil activation is not known, however, T cells are fundamental in their activation (28).
It is currently believed that the complex interaction between T cells, neutrophils and APC are involved in the immune pathogenesis of BD (hypersensitivity of T cells to different types of antigens; cytokines produced by T cells and APC causing neutrohil hyperactivation; cytokines secreted by activated neutrophils that contribute to their own activation and also stimulate Th1 cells (28).
The pathogenesis of BD is poorly understood. It is believed to be due to an autoimmune process triggered by external factors such as an infection, heat shock proteins (HSPs), neutrophil hyperfunction, increased in γδ T cells and pro-coagulatory factors in a genetically predisposed indivdual since the genetic contribution to the pathogenesis is estimated to be 20-30% (28). Figure 5 below illustrates and summarises the various hypotheses' that have been suggested for the pathogenesis of BD (28).
Figure 5: This figure shows the hypothesis of the various pathways of pathogenesis of BD. (1) Genetic factors, e.g. HLA-B*51, induce a general hyperactivity of the immune system (TH1-response,
granulocytes). (2) Bacterial or viral infection stimulate the expression of HSP60 (self) and MICA on different cells (e.g. endothelial cells), upregulation of adhesion molecules, activation of coagulation, stimulation of T cells (esp. gd and NK cells), continuing elevation of the cytokine production and finally induce (3) tissue damage by vasculitis. Additionally, a molecular mimicry with HLA-B*51 may play a role. In parallel, B-cells will be stimulated polyclonally (not shown on the figure) and may produce more antibody (e.g. against HSP).
4.5 Gene Associations with BD
4.5a HLA typing and PTPN22 620W polymorphism with Behcet's disease
BD has been associated with MHC class 1 complex molecules (25). HLA-B51 allele located in the MHC locus, on chromosome 6p has been the most strongly associated risk factor for BD in areas such as Turkey and Japan in comparison to Caucasians. HLA-B51 restricted cytotoxic T cell, which is autoreactive to MICA are found in active BD patients. HLA-B51 causes a hyperactivation of the immune system (23,28). Since HLA-B51 is the strongest disease associated gene in populations with Behcet's disease there may be a link between a reduced prevalence of PTPN22 R620W and an increased prevalence of HLA-B51 as both confer increased susceptibility to TB, an association further supported by the finding of heightened specific immunological responses to mycobacterial heat shock proteins in patients with BD (30). Since many of these responses are mediated through elements of the innate immune system (NK cells and T cells) which are upregulated in active Behcet's disease, it would be reasonable to hypothesise that BD may be a mycobacterially driven auto-inflammatory process as opposed to an autoimmune one. However, it is clear that PTPN22 620W is not the only predisposing factor in BD and that the complex interaction of infectious agents, genetic polymorphism and immune responses has yet to be elucidated (2).
Several other genes, located outside the MHC region have been proposed to be involved in BD pathogenesis, namely genes of IL-1, coagulation factor V, ICA M-1, TNFα gene (31), closely linked to HLA-B51, and endothelial nitric oxide synthetase (eNOS). Recent studies point that Mediterranean Fever gene (MEFV) mutations are an additional genetic susceptibility factor in BD (28).
4.6 Environment (infectious agents, heat shock proteins) and self-antigens
Proof that environment plays a role: when individuals from endemic areas who have immigrated to areas with low prevalence of the disease have an intermediate risk for developing the disease. The most generally accepted theory for the role of infectious agents is that microorganism antigens have high homology with human proteins (like heat shock protein (HSP) 65, obtained from Mycobacterium, which has high homology with human protein HSP60) and that cross-reaction leads to immune response.
Treatment of BD ranges from immunosuppressive drugs such as corticosteroids for posterior uveitis, Cyclosporin A, which is more commonly used, Azathioprine, Colchicine (also used in FMF) and many others. Biologicals including TNF-α antagonists and IFN-α (infliximab), which are monoclonal antibodies directed against inflammatory mediators or their receptors are used in treatment for BD (28).
All the diseases ranging from autoinflammatory (Crohn's disease), mixed pattern diseases (BD and Ankylosing spondylitis) and autoimmune diseases (T1D, RA, SLE, MS) have been associated with HLA genetic factors. A pooled analysis of MHC associations in the autoimmune diseases identified predisposing variants such as HLA-DRB1. HLA-DR2 and HLA-DR3 have been found to be predisposed in MS and SLE.
Genes such as CTLA4 and PTPN22 are associated with multiple autoimmune diseases and this suggests that various immunological pathways are common to multiple autoimmune diseases, whereas other pathophysiological mechanisms are specific to a particular disease. In contrast, these genes are not associated with MS suggesting that MS has a different pathogenesis pathway.
Another difference between organ-specific autoimmune diseases such as RA and T1D compared to MS is that the literature usually concentrates on tolerance, immune cells and other aspects of the immune system for RA and T1D. However, in MS, the role of the CNS in targetting the disease process and interaction with the immune system has been considered. This shows that genetic associations alone cannot be used to classify these diseases. Other factors need to be taken into account such as epidemiology, pathogenesis and treatment.
FMF has been associated Behcet's disease by the MEFV mutation. The epidemiology of the two diseases is along the 'Silk route' and the highest prevalence is seen in Turkey. Both of the diseases show hyperactivity of neutrophils and vasculitis and treatment involves Colchicine hence this may be sufficient evidence to prove BD has an autoinflammatory nature. Additional evidence supporting this link to autoinflammatory diseases is that the pathophysiology of BD is characterised by immune hyper-reactivity mainly affecting elements of the innate immune system and it is associated with pathogens such as bacteria and viruses leading to infections on the mucosal surfaces.
CTLA-4 polymorphism responsible for downregulating T cell activation has been shown to be associated with ocular disease but not directly with BD. However, PTPN22 R620W would be associated with BD as it is widely considered to be an autoreactive disease, although it has never been linked to specific autoantibody production. PTPN22 R620W has been associated with susceptibility to Gram positive bacterial infections consistent with a protective role for T cells in the early immune response to pneumococcal infection. BD has been aetiologically linked to the presence of a variety of Gram positive bacteria resident at mucosal surfaces, in particular streptococcus and Staphylococcus species. (28,29) This provides evidence that BD is a mixed pattern disease as it has features (eidemiology and treatment) associated with an autoinflammatory disease (FMF) and the clinical manifestations (posterior uveitis) and environmental trigger (pathogen) of BD are associated with CTLA-4 and PTPN22 genes respectively.