**2. Autoantibodies: a brief overview**

## **2.1. Autoantibody classes**

Antibodies are secreted heterodimeric proteins comprising light and heavy chains which are produced in mammals through recombination of V(D)J segments in developing B-lymphocytes. At any one time, there are thought to be of the order of 10<sup>7</sup> –10<sup>8</sup> different antibody sequences present in human serum. In response to the presence of foreign antigens or pathogens, somatic hypermutation processes drive the affinity maturation of specific antibody sequences, resulting in the production of high affinity, antigen-specific antibodies. Affinity-matured antibodies, or immunoglobulins (Igs), are produced by plasma cells and secreted into the blood stream where they scavenge their cognate antigen for destruction. Antibodies thus play a crucial adaptive role in mammalian defence mechanisms against harmful components that can cause disease. There are five classes of antibodies: IgG, IgM, IgE, IgD and IgA, which differ in their structures and immune functions. IgG is the major antibody class found in blood, has the longest serum half-life of all immunoglobulin isotypes [2] and contributes directly to a neutralising immune response to extracellular pathogens and toxins. IgA is also involved in direct neutralisation of toxins, virus and bacteria; however, it concentrates particularly in mucosal surfaces. IgM, a pentameric immunoglobulin, is the largest of the antibody classes and is associated with a primary immune response; IgMs are therefore frequently used to diagnose acute exposure to an immunogen or pathogen [2]. IgD and IgE are found in trace amounts in the blood with short half-lives. IgD remains membrane-bound and is involved in regulation of cell activation while IgE is associated with hypersensitivity and allergic reactions [2]. Classical autoantibodies are typically IgMs and include: anti-nuclear antibodies (ANA), which bind to the nuclear membrane, nucleoplasm, nucleoli and nuclear organelles of cells [3]; rheumatoid factor (RF), which binds with relatively low affinity to the Fc region of IgGs and which is found in the serum of rheumatoid arthritis (RA) patients [4]; Anti-double-stranded DNA (dsDNA) antibodies, anti-Sm antibodies, antiphospholipid antibodies, anti-Ro, anti-ribonucleoprotein and anti-La Antibodies which are all frequently found in systemic lupus erythematosus (SLE) patients [4]; and Anti-Sjogren's syndrome A (SSA) and -B (SSB) antibodies, which are found in many patients with Sjogren's syndrome [4].

### **2.2. Causes of autoantibody production**

referred to as autoimmunity. Classic autoimmune syndromes include systemic lupus erythematosus, rheumatoid arthritis, rheumatic heart disease, Graves' disease, autoimmune hepatitis, multiple sclerosis, diabetes, and Sjogren's syndrome. In such autoimmune diseases, it is well established that the pathology relates directly to the autoantibodies. However, it is increasingly recognised that autoantibodies are also found in many other diseases, including cancers, cardiovascular diseases, neurodegenerative diseases, as well infectious diseases such as malaria, albeit in such diseases it is not yet clear whether the autoantibodies play a direct role in the pathology or whether they are merely symptomatic of disease. Irrespective of whether the autoantibodies are causative or symptomatic of specific diseases though, there is increasing interest globally in exploring the clinical potential of circulating autoantibodies as diagnostic biomarkers and considerable research effort is now being directed to the discovery, quantitation and validation of novel autoantibody-based diagnostic biomarkers in many

Numerous techniques have been utilised over the last few decades to detect the presence of autoantibodies in patient samples, not least since autoantibodies are increasingly thought to represent excellent potential biomarkers for early disease detection. Techniques that have historically been employed for biomarker identification include western blotting, immunohistochemistry and enzyme-linked immunosorbent assays (ELISA), but these are being superseded now by newer technologies that offer higher multiplicity as well as greater sensitivity and specificity. Amongst these newer technologies, protein microarrays are becoming established now as a powerful means to detect protein expression levels and to investigate protein-ligand interactions, as well as to probe protein function [1], since they enable efficient and sensitive, high throughput protein analysis, with large numbers of technically-replicated measurements being made in parallel using miniaturised assay formats and minimal sample volumes. These properties of protein microarrays make them ideally suited to component-resolve and quantify autoantibody profiles in biological

Antibodies are secreted heterodimeric proteins comprising light and heavy chains which are produced in mammals through recombination of V(D)J segments in developing

antibody sequences present in human serum. In response to the presence of foreign antigens or pathogens, somatic hypermutation processes drive the affinity maturation of specific antibody sequences, resulting in the production of high affinity, antigen-specific antibodies. Affinity-matured antibodies, or immunoglobulins (Igs), are produced by plasma cells and secreted into the blood stream where they scavenge their cognate antigen for destruction. Antibodies thus play a crucial adaptive role in mammalian defence mechanisms against

–10<sup>8</sup>

different

B-lymphocytes. At any one time, there are thought to be of the order of 10<sup>7</sup>

different disease areas.

160 Autoantibodies and Cytokines

samples.

**2. Autoantibodies: a brief overview**

**2.1. Autoantibody classes**

In a normal immune response to a foreign antigen, professional antigen presenting cells including dendritic cells, B-cells and macrophages – engulf and proteolyse the antigen and then present antigen-derived peptides on their cell surface in the form of major histocompatibility complexes; recognition of complexed peptides by a specific receptor on a T-cell then triggers the release of cytokines and chemokines, resulting in activation of that T-cell. Interaction between antigen-specific T- and B-cells subsequently leads to antigen-specific B-cell proliferation [1, 2]. A portion of those B-cells serve as memory cells, whilst the remainder act as effector cells that differentiate into antibody-producing plasma cells responsible for the production and release of antigen-specific antibodies [5].

Peripheral tolerance mechanisms usually ensure that self-reactive T- and B-cells (i.e. displaying T- or B-cell receptors for self-antigens) are suppressed. However, in certain circumstances, peripheral tolerance can be broken, resulting in proliferation of autoantigen-specific T- and B-cells. Simplistically, peripheral tolerance can be broken for a number of reasons, for example if the self-antigen is significantly over-expressed in a tissue or if neoantigens are somehow presented to the host immune system. Such neoantigens can include mutated peptide epitopes, aberrantly spliced or aberrantly post-translationally-modified epitopes, or new discontinuous epitopes resulting from misfolding of the antigen. Tolerance defects can also stem from the downregulation of regulatory T-cells (Tregs) [6], whilst chronic inflammatory responses are thought to facilitate the release and exposure of intracellular antigens to the immune system, resulting in autoantibody production in cancer patients [7], as well as increased vasculature permeability, allowing immune cell accumulation at the tumour site [8]. One consequence of loss of peripheral tolerance can be the production of self-antigenspecific autoantibodies.

As mentioned above, autoantigens may result from aberrant post-translationally modifications, including proteolysis, hydrolysis, phosphorylation and oxidation [9]. One such example occurs in RA, where patients produce autoantibodies against citrulline-modified proteins, themselves produced by the enzymatic action of peptidylarginine deiminases (PADs) - calcium-dependent enzymes that catalyse the post-translational hydrolysis of peptidylarginine to peptidylcitrulline. During inflammation, oxidative stress or apoptosis, PAD converts specific arginine residues on selected proteins into citrulline (a process often referred to as 'citrullination'), thereby producing neoepitopes that are recognised as non-self, dramatically altering immunogenicity and autoantibody production in RA patients [10].

Autoantibodies are also produced in response to the uncontrolled released of autoantigens during cell death processes. Maintenance of tissue homeostasis ordinarily takes place via clearance of apoptotic and altered cells through phagocytosis- or complement-dependent mechanisms, inhibition of inflammation, removal of misfolded proteins, and regulation of autoantibody-producing B cells [11]. However, when clearance mechanisms becomes compromised, dead cells accumulate and progress to secondary necrosis, releasing autoantigens as well as pro-inflammatory markers and thereby disrupting immune homeostasis [12] (**Figure 1**).

Autoantibody production thus has a multifactorial aetiology in which environmental and inherited factors interplay in determining the autoantibody profiles of an individual. Environmental factors associated with autoantibody production include drugs, toxins, chemicals from personal care products, and infections. Exposure to such agents can result in modification or mutation of chromosomal DNA sequences, potentially giving rise to altered gene- and protein expression (which can drive altered post-translational modifications), as well as to the expression of aberrantly-spliced or mutated form of proteins, all of which can result in the generation of neoantigens in the exposed tissue and hence to the production of specific autoantibodies. Furthermore, genetic predisposition or family history of autoimmune disorders also contributes to one-third of the risk of having increased autoantibody levels and various genome-wide association studies have shown that the production of autoantibodies in SLE [13], RA [14] and Multiple Sclerosis [15] is controlled by multiple loci.

**2.3. Gender bias in autoimmune diseases**

fetal micro-chimerism [20].

and severity of which vary between individuals [19].

**2.4. Functional role of autoantibodies in disease**

The term 'autoimmune disease' refers to a group of over 80 distinct disorders, the symptoms

**Figure 1.** Key factors that increase antigenic pressure and cause the production of autoantibody in several diseases.

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There are marked differences in diseases that predominantly affect males or females, as shown in **Figure 2**. Generally, females are more susceptible to autoimmune diseases whereas males show increased susceptibility to non-reproductive cancers. As females tend to have more responsive and robust immune system compared to their male counterparts, it is therefore not surprising that females respond more aggressively to autoantigens and are more susceptible to autoimmune diseases [20]. Other factors that contribute to the sex bias of autoimmune diseases include X-chromosomal abnormalities, X-chromosomal inactivation, and

The outcome of aberrant activation of the immune system and inflammatory process is dependent on multiple factors, including the type of affected tissue or organ and the degree of tissue injury sustained [21]. For example, in type 1 diabetes mellitus, the immune system reacts to insulin-producing cells in the pancreas. In other examples, tissues of the small intestines are

Although the self-reactivity of autoantibodies can be harmful to host tissues, recent studies suggest that low-grade self-reactivity also occurs in healthy individuals, implying that certain autoantibodies may play a role in maintaining immune homeostasis [16] and in protecting against pathogenic processes, by activating innate and acquired immunity to maintain or restore health status [16]. Natural autoantibodies are predominantly of the IgM class, which makes sense since IgM is the first antibody to appear when the immune system is triggered in response to external antigenic exposure. By contrast, circulating naive IgMs arise without known immune exposure or vaccination [11] but have also been reported to recognise certain autoantigens in healthy adults as well as in newborn babies [17, 18].

Autoantibody-Based Diagnostic Biomarkers: Technological Approaches to Discovery and Validation http://dx.doi.org/10.5772/intechopen.75200 163

**Figure 1.** Key factors that increase antigenic pressure and cause the production of autoantibody in several diseases.

### **2.3. Gender bias in autoimmune diseases**

increased vasculature permeability, allowing immune cell accumulation at the tumour site [8]. One consequence of loss of peripheral tolerance can be the production of self-antigen-

As mentioned above, autoantigens may result from aberrant post-translationally modifications, including proteolysis, hydrolysis, phosphorylation and oxidation [9]. One such example occurs in RA, where patients produce autoantibodies against citrulline-modified proteins, themselves produced by the enzymatic action of peptidylarginine deiminases (PADs) - calcium-dependent enzymes that catalyse the post-translational hydrolysis of peptidylarginine to peptidylcitrulline. During inflammation, oxidative stress or apoptosis, PAD converts specific arginine residues on selected proteins into citrulline (a process often referred to as 'citrullination'), thereby producing neoepitopes that are recognised as non-self, dramatically

Autoantibodies are also produced in response to the uncontrolled released of autoantigens during cell death processes. Maintenance of tissue homeostasis ordinarily takes place via clearance of apoptotic and altered cells through phagocytosis- or complement-dependent mechanisms, inhibition of inflammation, removal of misfolded proteins, and regulation of autoantibody-producing B cells [11]. However, when clearance mechanisms becomes compromised, dead cells accumulate and progress to secondary necrosis, releasing autoantigens as well as pro-inflammatory markers and thereby disrupting immune homeostasis [12]

Autoantibody production thus has a multifactorial aetiology in which environmental and inherited factors interplay in determining the autoantibody profiles of an individual. Environmental factors associated with autoantibody production include drugs, toxins, chemicals from personal care products, and infections. Exposure to such agents can result in modification or mutation of chromosomal DNA sequences, potentially giving rise to altered gene- and protein expression (which can drive altered post-translational modifications), as well as to the expression of aberrantly-spliced or mutated form of proteins, all of which can result in the generation of neoantigens in the exposed tissue and hence to the production of specific autoantibodies. Furthermore, genetic predisposition or family history of autoimmune disorders also contributes to one-third of the risk of having increased autoantibody levels and various genome-wide association studies have shown that the production of autoantibodies in SLE [13], RA [14] and Multiple Sclerosis [15] is controlled by

Although the self-reactivity of autoantibodies can be harmful to host tissues, recent studies suggest that low-grade self-reactivity also occurs in healthy individuals, implying that certain autoantibodies may play a role in maintaining immune homeostasis [16] and in protecting against pathogenic processes, by activating innate and acquired immunity to maintain or restore health status [16]. Natural autoantibodies are predominantly of the IgM class, which makes sense since IgM is the first antibody to appear when the immune system is triggered in response to external antigenic exposure. By contrast, circulating naive IgMs arise without known immune exposure or vaccination [11] but have also been reported to recognise certain

autoantigens in healthy adults as well as in newborn babies [17, 18].

altering immunogenicity and autoantibody production in RA patients [10].

specific autoantibodies.

162 Autoantibodies and Cytokines

(**Figure 1**).

multiple loci.

The term 'autoimmune disease' refers to a group of over 80 distinct disorders, the symptoms and severity of which vary between individuals [19].

There are marked differences in diseases that predominantly affect males or females, as shown in **Figure 2**. Generally, females are more susceptible to autoimmune diseases whereas males show increased susceptibility to non-reproductive cancers. As females tend to have more responsive and robust immune system compared to their male counterparts, it is therefore not surprising that females respond more aggressively to autoantigens and are more susceptible to autoimmune diseases [20]. Other factors that contribute to the sex bias of autoimmune diseases include X-chromosomal abnormalities, X-chromosomal inactivation, and fetal micro-chimerism [20].

### **2.4. Functional role of autoantibodies in disease**

The outcome of aberrant activation of the immune system and inflammatory process is dependent on multiple factors, including the type of affected tissue or organ and the degree of tissue injury sustained [21]. For example, in type 1 diabetes mellitus, the immune system reacts to insulin-producing cells in the pancreas. In other examples, tissues of the small intestines are

aggregates in Progressive Supranuclear Palsy (PSP), a rare disease often misdiagnosed as Parkinson's disease. In AD, Tau causes misfolding of beta-amyloid, leading to amyloid-β (Aβ) plaque formation and downstream pathology, but in PSP, Tau itself mis-folds and agglomerates. These protein agglomerations subsequently leave the cell, spread throughout the brain and disrupt the communication between neurons [25]. Interestingly, a recent study identified an anti-Aβ plaque autoantibody in certain aged but cognitively firm individuals that was absent in AD patients; this autoantibody was cloned and has been shown to selectively target aggregated Aβ in a mouse model of AD, where it bound parenchymal Aβ and reduced soluble and insoluble Aβ in a dose-dependent manner; in Phase 2 clinical trials, this autoantibody, Aducanumab, reduced brain Aβ in patients with mild AD, again in a dose-dependent manner [26], strongly suggesting that anti- Aβ autoantibodies play a protective role in healthy

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165

In cancers, chronic inflammation is a well-recognised hallmark and it is known that both cancer and autoimmune diseases can occur in the same individual, albeit in cancer, the immune response is often suppressed and unable to eliminate altered self-cells, while in autoimmune diseases it is hyper-activated against specific autoantigens. The act of manipulating the immune system in different ways, however, suggests a possible link between these two conditions [21] and it seems likely that inflammatory processes drives both autoimmunity and malignancy. However, it remains unclear whether it is the underlying autoimmunity that leads to malignancy ("inflammation-induced cancer") or whether the immune responses directed against tumour antigens lead to autoimmune diseases ("tumour-induced

Autoantibody production is a key indicator of many diseases and has emerged as an important tool in predicting onset of a number of diseases. Autoantibodies are in principle detectable many years before manifestation of disease or symptoms and have been observed in an ever-widening range of disease areas, which makes novel autoantibodies attractive plausible biomarkers for early diagnosis of a broader spectrum of diseases now. Known autoantibody biomarkers have been reported to predate symptoms in Sjogren's syndrome, rheumatoid

Sjogren's syndrome is an autoimmune disease that affects parts of the body which produce secretions such as tears and saliva. The symptoms overlap with other autoimmune conditions and can range from mild to severe, causing nausea, fatigue, joint pain as well as excessive dryness of the eyes and mouth. Autoantibodies attack cells in mucous membranes and moisture-secreting glands of the eyes and mouth, causing dryness, irritation and pain. A study published in 2015 concluded that autoantibodies are present up to 18–20 years before the diagnosis of primary Sjogren's syndrome [27]. A total of five autoantibodies were analysed, namely antinuclear antibodies, rheumatoid factor and autoantibodies against Ro 60/SSA, Ro

**3. Diagnostic utility of autoantibody biomarkers**

arthritis, Alzheimer's disease and cancers, as discussed below:

individuals.

autoimmunity").

**3.1. Early detection of disease**

**Figure 2.** Gender-dependent susceptibility to autoimmune diseases, infectious diseases, inflammatory diseases and cancers.

affected in inflammatory bowel disease, while myelin - a fatty substance that protects nerve fibres in the brain and spinal cord - is destroyed in Multiple Sclerosis. In RA, connective tissues are affected and in SLE, auto-reactivity usually occurs in skin, heart and lung tissues. Sjogren's syndrome occurs when autoantibodies target secretory glands that produce tears and saliva, causing extreme dryness and other complications [22].

In other diseases, however, the functional role of autoantibodies is less clear. For example, in neurodegenerative diseases such as Parkinson's and Alzheimer's Diseases, increased cellular toxicity is caused by the accumulation and aggregation of misfolded proteins, which might also result in the generation of protective autoantibodies in some patients. For example, in Parkinson's Disease (PD), the protein alpha-synuclein misfolds and aggregates to form Lewy bodies; these bodies form in the brain tissues of PD patients and infiltrate the neurons, disrupting signalling process in the brain. A recent study reported that a defined set of epitopes derived from alpha-synuclein drive cytotoxic T-cell responses in people with PD [23], whilst another recent study reported a decline in anti-alpha-synuclein autoantibodies in PD patients compared to controls, suggesting that in some patients anti-alpha-synuclein autoantibodies might play a protective role [24].

Similarly, in Alzheimer's Disease (AD), the microtubule-associated protein Tau accumulates and aggregates in neurons causing neuronal degeneration. Tau also accumulates and aggregates in Progressive Supranuclear Palsy (PSP), a rare disease often misdiagnosed as Parkinson's disease. In AD, Tau causes misfolding of beta-amyloid, leading to amyloid-β (Aβ) plaque formation and downstream pathology, but in PSP, Tau itself mis-folds and agglomerates. These protein agglomerations subsequently leave the cell, spread throughout the brain and disrupt the communication between neurons [25]. Interestingly, a recent study identified an anti-Aβ plaque autoantibody in certain aged but cognitively firm individuals that was absent in AD patients; this autoantibody was cloned and has been shown to selectively target aggregated Aβ in a mouse model of AD, where it bound parenchymal Aβ and reduced soluble and insoluble Aβ in a dose-dependent manner; in Phase 2 clinical trials, this autoantibody, Aducanumab, reduced brain Aβ in patients with mild AD, again in a dose-dependent manner [26], strongly suggesting that anti- Aβ autoantibodies play a protective role in healthy individuals.

In cancers, chronic inflammation is a well-recognised hallmark and it is known that both cancer and autoimmune diseases can occur in the same individual, albeit in cancer, the immune response is often suppressed and unable to eliminate altered self-cells, while in autoimmune diseases it is hyper-activated against specific autoantigens. The act of manipulating the immune system in different ways, however, suggests a possible link between these two conditions [21] and it seems likely that inflammatory processes drives both autoimmunity and malignancy. However, it remains unclear whether it is the underlying autoimmunity that leads to malignancy ("inflammation-induced cancer") or whether the immune responses directed against tumour antigens lead to autoimmune diseases ("tumour-induced autoimmunity").
