Psychoneuroimmunology and Genetics

*Rama P. Vempati and Hemakumar M. Reddy* 

## **Abstract**

 Psychoneuroimmunology is a study that investigates the interaction between human emotions and the immune system, which is mediated by the endocrine and nervous systems. The nervous and immune systems maintain extensive communication, including communication to lymphoid organs from deep-rooted sympathetic and parasympathetic nerves. Genetic factors are responsible for individual variation in emotional reactivity, and neuroendocrine stress responses were shown by earlier studies in humans. Several gene-environment studies have shown that long-term effects of stress are being moderated by genetic variations in the hypothalamic-pituitary-adrenal (HPA) axis. There is a large interindividual variability of HPA axis stress reactivity on variants of the glucocorticoid (GR) or mineralocorticoid receptor genes, and it documents a sex-specific association between different GR gene polymorphisms and salivary cortisol responses to acute psychosocial stress. In conclusion, many kinds of mind-body behavioral interventions are effective in improving mood, quality of life, reducing stress, and anxiety, thereby altering neuroendocrine and immune functions, and ultimately altering the genetic aberrations. However, the question remains as to whether these latter effects are sufficiently large or last long enough to contribute to health benefits, or if they are even relevant to the development of a disease.

**Keywords:** psychoneuroimmunology, immunology and genetics, emotional stress, genetic factors involved in stress, epigenetics involved in stress

### **1. Psychoneuroimmunology**

Psychoneuroimmunology is an area that examines the interaction between human emotions and the immune system, which is mediated by the endocrine and nervous system. The brain controls the immune system by hardwiring sympathetic and parasympathetic nerves to lymphoid organs. Further neuroendocrine hormones such as a corticotropin-releasing hormone or substance P regulate cytokine balance. The immune system controls some brain activities such as sleep and body temperature. Based on anatomical and a close functional connection, the nervous and immune systems act in a very mutual way. Over recent decades, reasonable evidence has emerged that these brain-to-immune interactions are highly modulated by psychological factors which influence immunity and immune system-mediated disease [1].

The nervous and immune systems maintain extensive communication, including communication to lymphoid organs from deep-rooted sympathetic and

parasympathetic nerves. Acetylcholine, norepinephrine, vasoactive intestinal peptide, substance P, and histamine such as neurotransmitters modulate immune activity. Corticotropin-releasing factor, leptin, and alpha-melanocyte-stimulating hormone such neuroendocrine hormones regulate cytokine balance. The brain activity mainly body temperature, sleep, and feeding behavior is influenced by the immune system. The major histocompatibility complex directs T cells to immunogenic molecules held in its cleft and also controls the development of neuronal connections. Neurobiologists and immunologists are exploring common ideas like the synapse to understand properties such as memory which is shared between these two systems [2].

Both neuronal (direct sympathetic innervation of the lymphoid organ) and neuroendocrine (hypothalamic-pituitary-adrenal axis) pathways are involved in the control of the humoral and cellular immune responses. There is a recent evidence on the immunosuppressive effect of acetylcholine-secreting neurons of the parasympathetic nervous system which influences the central nervous system primarily through cytokines. Neuroimmune signal molecules such as hormones, neurotransmitters, neuropeptides, cytokines, or their receptors enable mutual neuroimmune communication. Subcellular and molecular mechanisms of cytokine-neuropeptide/neurotransmitter interactions were extensively investigated. At the neuroanatomical level, neuroimmune communication in the role of discrete brain areas related to emotionality has been established. Immuno-enhancement, including the antitumor cytotoxic activity and antiviral activity, related to the "brain reward system," limbic structures, and neocortex, offers a new direction for therapy in immune disorders [3].

#### **2. Immunology and genetics**

Genetic predisposition is important for this immune function. Stress-mediated inflammation is a common feature of many hereditary disorders, due to the proteotoxic effects of mutant proteins. Harmful mutant proteins can induce dysregulated IL-1β production and inflammation. Depressive disorders are often accompanied by profound changes in immunity. Clinical observations in depression disorders showed that immune dysfunction is the main cause of increased risks in other oncological, inflammatory, and infectious diseases. Immunological reactions in psychoemotional stress play an important role. Studying Antidepressant-Sensitive Catalepsy (ASC) in mice showed a decrease in IgM immune responses and sensitivity to the administration of antidepressants. Unlike their non-depressive parental CBA strains, ASC lines show the difference in T-lymphocyte distribution and changes in IgG and IgM immune responses, low antibody production, abnormal CD4+ T-cell content in blood and spleen, and variations in CD4+/CD8+ T-cell ratio [4].

Stress-induced inflammation is a key pathogenic factor in inherited diseases and autoinflammatory syndromes. The stress contributes severity of the symptoms in these diseases. A study showed the correlation among basal stress, disease severity, and antioxidant response in two different cryopyrin-associated periodic syndrome (CAPS) patients sharing same nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing 3 (NLRP3) mutation [5]. Hence, similar stressrelated mechanisms may operate in other genetic diseases, where inflammation causes disease progression and mutant protein present in monocytes. Improving the responses to stress represents a promising therapeutic opportunity for this kind of serious diseases, while considering the genetic factor (individual tolerance levels) may play a major role.

## **3. Molecular mechanisms of emotional stress**

Identification of mechanisms underlying a dysregulation of major components of the stress response system is a very challenging task as it involves complex cellular interactions at the level of different organs and systems. One of the main features of the stress response is the activation of the hypothalamic-pituitaryadrenal axis (HPA) [6]. The main regions of the brain that shows stress response are hippocampus, amygdala, and prefrontal cortex. Decreased activity and neuronal atrophy in the hippocampus and in the prefrontal cortex, as well as increased activity and neuronal growth in the amygdala, are involved in post-traumatic stress disorder (PTSD) [6]. The changes that stress induces mainly affect the levels of cortisol and catecholamines (epinephrine, norepinephrine, dopamine). Catecholamines are released shortly after stress onset and go back to normal levels upon stress termination. Glucocorticoids act by binding to two types of receptors—mineralocorticoid receptors (MR) and glucocorticoid receptors (GR). Molecular mechanisms involving this stress response are genetic, epigenetic, and immunological nature.

## **4. Hormonal and immunological factors in stress response**

 The primary hormonal end product of the HPA axis is cortisol. A longitudinal study of 358 Dutch adolescents with a mean age of 15 years over 3 years showed that cortisol awakening response (CAR) moderated the effects of depressive symptoms on violent adult outcomes. The results showed that depressive symptoms were positively associated with violent outcomes when CAR levels are low [7].

Mental and physical stress can suppress the immune system in both humans and animals. Chronic stress-induced alterations in immune responses could result from increased cell death and apoptosis or decreased cell proliferation. It is well known that exhausting physical activity and mental stress lead to immunosuppression of the immune system by steroid hormone regulation. Chronic stress significantly enhances corticosterone production and induces lymphocyte apoptosis [8, 9]. Stress hormones like cortisol play a fundamental role in regulating immune responses and the balance of T helper (Th) 1 and Th2 cytokines, thereby modulating the susceptibility of various immune-related disorders. Toll-like receptors (TLRs) play a key role in modulating immune responses, cell apoptosis, and cell survival. Among 11 known TLRs in mammals, TLR9 plays a major role in chronic stress-induced immune suppression by modulating corticosteroid levels [10].

Psychosocial stressors increase peripheral cytokine production, a potentially important factor in the development of depression or anxiety [11, 12]. Subsets of patients with the major depressive disorder (MDD) and post-traumatic stress disorder have higher levels of multiple inflammatory markers, including the cytokine interleukin 6 (IL-6) [11, 13]. Preexisting differences in the sensitivity of an individual's peripheral immune system like cytokine interleukin 6 (IL-6) dictates their subsequent vulnerability or resilience to social stress [14].

## **5. Genetic factors involved in stress**

 Molecular studies of the stress phenomenon have found some genes which are differentially expressed in stressed individuals and control subjects. Studies involving effect at individual genes as well as genome-wide studies at cellular, tissue, and individual levels are reported.

A study of DNA microarray from circulating leucocytes showed that the stress causes some genes upregulated and some other genes downregulated. The downregulated genes are mainly related to apoptosis, cell cycle inhibitors, NF-KB inhibitor (Apo J), and antiproliferative cytokines. The upregulated genes are involved in cell cycle activation, and enzymes involved in nucleic acid biosynthesis and proteins. Other upregulated genes are transcription factors that control chromatin structure and cell growth [15]. The transcription factor that controls many of these genes is NF-KB. Hence, NF-KB plays a key role in the cellular stress response.

Researchers have attempted to attribute genetic variation among individuals to their neuroendocrine responsiveness to environmental stimuli like stress by studying how the immune system interacts with the nervous and endocrine systems and, together, how they impact upon the course and outcome of disease [16]. As early as 1992, Gatz et al. studied the importance of genes and environments on the symptoms of depression [17].

Genetic factors are responsible for individual variation in emotional reactivity, and neuroendocrine stress responses were shown by family and twin studies in humans and by the study of inbred strains and selection experiments in animals [18]. A twin study revealed the significant genetic impact on the cortisol awakening response with heritability estimates between 0.40 and 0.48 for the mean cortisol increase after awakening and the area under the curve, respectively [19]. An increased cortisol awakening response in individuals reporting chronic stress includes social stress and lack of social recognition [19].

Several gene-environment studies have shown that long-term effects of stress are being moderated by genetic variations in the hypothalamic-pituitary-adrenal (HPA) axis. Studies by Wust et al. investigated contribution of large interindividual variability of HPA axis stress reactivity on variants of the glucocorticoid or mineralocorticoid receptor genes and documented a sex-specific association between different GR gene polymorphisms and salivary cortisol responses to acute psychosocial stress [20]. Single-nucleotide polymorphisms (SNPs) associated with stress vulnerability and resilience are found in the GR (e.g., through regulation by the FK506 binding protein 5 [FKBP5]), the corticotropin-releasing hormone factor receptor 1 (CRHR1), and the MR genes [21].

 The genetic component is often complex in these studies and involves several genes and, hence, should study the quantitative trait loci (QTL). It is easy to study QTL in plants and animals where you can easily get the inbred lines where the genetic makeup is similar among individuals. Quantitative genetic analysis to behavioral responses to environmental challenges like stress in humans is done mainly on the large cohorts of families and twins.

Another approach is the utilization of genome-wide association studies (GWAS) that would facilitate identification of new genes involved in stress development and elucidate the molecular pathways which are dysregulated. In contrast to candidate gene studies that are based on prior biological knowledge, in GWASs common variants across the whole genome are screened concerning the contributing genes. GWASs for human stress-related phenotypes are rare [21]. A meta-analysis on plasma cortisol levels in 12,597 participants found a genome-wide association of SNPs in the SERPINA6/SERPINA1 locus. GWAS and individual gene studies are often underpowered owing to smaller sample sizes. There is a need to test whether the identified candidate genes appear to be nominally significant in the GWASs in larger samples [21].

## **6. Epigenetics involved in stress**

Even though genome is the blueprint for biological activity, the epigenome adds another layer on top of the genome and serves to modulate gene expression in response to environmental cues. Epigenetic modification induced by environmental factors could influence the development of chronic pain by modulating genomic expression of one or more biological systems associated with pain and psychological stressors. Recent studies demonstrate that adverse psychosocial environments like stress can affect gene expression by altering the epigenetic pattern of DNA methylation, chromatin structure by histone modifications, and noncoding RNA expression [22].

 Most of the epigenetic studies employ animal models at early life experiences that demonstrate epigenetic modification that occurs in response to stressors, which alter the developing epigenome in the hippocampus. Some studies evaluate epigenetic modification using adult models of stress and depression as well as consideration of the role of epigenetics in resilient versus susceptible phenotypes. Adverse events such as stress or maltreatment at early stages of development can more readily trigger epigenetic alterations which can adversely affect physiological function and behavior in adult life. Studies involving human samples from different models like suicide victims with and without child abuse, prenatal depression, and posttraumatic stress disorder showed altered DNA methylation patterns at the glucocorticoid receptor gene (NR3C1) [22]. The salivary cortisol response which in turn leads to altered central regulation of the HPA axis consequent to maternal depressed mood. Epigenetic changes due to stress affected the gene expression of several genes including estrogen receptor alpha (ER-A), trichostatin (TSA), N-methyl-daspartate (NMDA), nerve growth factor-inducible protein-A (NGFI-A), arginine vasopressin (AVP), brain-derived neurotrophic factor (BDNF), and cyclic-AMP response element-binding protein (CREB) [22]. Telomere shortening is one of the molecular indicators as an epigenetic effect on stress and chronic pain [23].

## **7. Biomarkers for diagnosis and treatment of stress**

 The hypothalamus-pituitary-adrenal axis (HPA axis) is a vital part of the human stress response system. The endocrine marker cortisol is a useful index of HPA axis activity, and it shows good intraindividual stability across time and appears to uncover subtle changes in HPA regulation. Cortisol activity and the response are important biological indicators of emotional and behavioral responses to environmental stressors. Low cortisol activity is hypothesized to be linked to antisocial behaviors [24]. Several studies demonstrated the role of age and gender; endogenous and exogenous sex steroid levels; pregnancy, lactation, and breastfeeding; smoking, coffee, and alcohol consumption; as well as dietary energy supply in salivary cortisol responses to acute stress [25]. Salivary cortisol levels are a reliable measure of HPA axis adaptation to stress and hence are a useful and valid biomarker in stress research [26].

 The knowledge of the molecular bases of genetic variability points to the biochemical pathways responsible for the differences in stress responses will allow the development of new therapeutic strategies for pathological conditions [18]. Interventions aimed at manipulating the epigenome are a real and promising possibility to circumvent the stress-related psychoneuroimmunology disorders. Epigenetic and telomere changes may offer an array of targets that can be exploited for prevention and treatment interventions [27].

#### *Immunogenetics*

In conclusion, many kinds of mind-body behavioral interventions are effective in improving mood, quality of life, reducing stress, and anxiety, thereby altering neuroendocrine and immune functions, and ultimately altering the genetic aberrations. However, the question remains as to whether these latter effects are sufficiently large or last long enough to contribute to health benefits or if they are even relevant to the development of a disease. Unfortunately, there is no strong body of evidence that supports the clinical correlation between psychoneuroimmunology and genetics and reaping the health benefits through behavioral interventions.

## **Author details**

Rama P. Vempati1 \* and Hemakumar M. Reddy2

1 Clinical Safety Sciences, Sunnyvale, CA, USA

2 Department of Molecular Biology, Cell Biology and Biochemistry, Brown University Division of Biology and Medicine, Providence, RI, USA

\*Address all correspondence to: rpvempati@gmail.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Psychoneuroimmunology and Genetics DOI: http://dx.doi.org/10.5772/intechopen.82557* 

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## **Chapter 4**

## Immunoassay Techniques Highlighting Biomarkers in Immunogenetic Diseases

*Emilia Manole, Alexandra E. Bastian, Ionela D. Popescu, Carolina Constantin, Simona Mihai, Gisela F. Gaina, Elena Codrici and Monica T. Neagu* 

## **Abstract**

Diagnosis of autoimmune diseases is crucial for the clinician and the patient alike. The immunoassay techniques most commonly used for this purpose are immunohistochemistry, ELISA, and Western blotting. For the detection of more specific biomarkers or the discovery of new ones for diagnostic purposes and as therapeutic targets, microarray techniques are increasingly used, for example, protein microarray, Luminex, and in recent years, surface plasmon resonance imaging. All of these technologies have undergone changes over time, making them easier to use. Similar technologies have been invented but responding to specific requirements for both diagnostic and research purposes. The goals are to study more analytes in the same sample, in a shorter time, and with increased accuracy. The reproducibility and reliability of the results are also a target pursued by manufacturers. In this chapter, we present these technologies and their utility in the diagnosis of immunogenetic diseases.

**Keywords:** immunoassay, protein biomarkers, autoimmune diseases, IHC, ELISA, WB, protein microarray, SPRi, Luminex

### **1. Introduction**

An autoimmune pathology occurs when the immune system loses its ability to distinguish between its own cells and nonself cells, inducing the attack of selftissue. This mechanism involves both the environmental factors and the genetic predisposition of the individual.

Proteomic technologies identify and separate different proteins of interest from biological samples, thus enabling their characterization as biomarkers, establishing their interactions, their role and the mechanisms in which they are involved, the identification of new diagnostic and therapeutic targets. The identification of protein biomarkers may be the basis for developing new methods of early diagnosis and treatment [1]. In general, an ideal biomarker should meet certain characteristics: be specific to a particular disease, be validated and confirmed as having specificity for that pathology, be able to early identify the disease, its testing to be easy and cheap as far as possible, reliable, and noninvasive [2, 3].

#### *Immunogenetics*

Although important advances have been made in deciphering immune function, the understanding of this function dysregulation and the specific autoimmune response remains limited. The domain is complex and includes, besides the disturbance of immune system functioning, gene alterations that regulate and control the self-tolerance. In this chapter, we will describe the techniques of highlighting the proteomic biomarkers involved in the pathogenesis of immunogenetic diseases.

In the case of immunogenetic diseases, one of the tissues that are first tested for specific biomarkers is blood, namely, the serum, which contains approximately 60–80 mg/mL proteins, besides amino acids, lipids, salts, and carbohydrates [4]. Applying proteomic immunoassay techniques for the diagnosis of immunogenetic diseases may also predict the course of disease, or result in a personalized treatment for patients [5, 6].

Proteomic biomarkers are particularly useful for providing the information on cellular signaling pathways, bringing early disease data, monitoring treatment response or adverse effects. They can be monitored from body fluids other than blood, such as: urine, saliva, cerebrospinal fluid and from different tissues (biopsies) [7].

The necessity to analyze very small amounts of proteins present in biological samples [8], as well as the increase in the number of proteins requiring simultaneous, reliable, reproducible, and significant investigations led to the modernization of the existing techniques and to the appearance of some new methods of biomarker investigation and analysis. Immunohistochemistry, ELISA, and Western blotting are of the old methods that changed, adapted, modernized over time, but remained "on barricades" for protein biomarker investigation, especially in autoimmune disorders. Immunoassay methodologies are the most commonly used tools in protein research, using the properties of antibodies to bind different protein domains and to mark them. Next, the methods abovementioned are the other high sensitivity technics for validating proteomic biomarkers such as protein microarray, surface plasmon resonance, and Luminex multiplex assays. In recent years, many multiplexed immunodetection techniques have been developed to simultaneously investigate multiple proteins (from several tens to several hundreds), in the same sample, and which are in very low amounts (**Figure 1**).

#### **Figure 1.**

*The schematic representation of the immunoassay methods presented in this chapter, more or less in the order in which they appeared in time and how they evolved. These methods are based on the protein/antigenantibody reaction that is shown on the left side—here is the indirect method: antigen* → *primary antibody* → *secondary antibody conjugated with a fluorochrome or an enzyme.* 

In many cases, the immunoassay techniques are used in conjunction for diagnostic, to confirm the presence of autoantibodies and then to characterize the expression of one or more specific biomarkers for a certain disease. More of this, these techniques can validate their mutual results.

## **2. Immunohistochemistry**

 The technique of immunohistochemistry (IHC) is a basic one, both in the anatomopathological diagnosis and in the research. It allows viewing of a protein of interest in a tissue section, specifying its location. This last aspect is very important and distinct IHC from other immunodetection techniques. The presence, reduction or the absence of the target protein allows a precise diagnosis or a personalized one. We do not intend to describe the technique itself, but we would like to mention it as the method of identifying immune antigens of interest, including immunogenetic diseases.

Based on the principle of the antigen-antibody reaction, this technique has undergone improvements over time. It started with a *direct IHC method*, the reaction antigen (target protein)-antibody, coupled with a fluorochrome. The first data on an attempt to use the direct IHC are from 1934 [9], but the use of fluorochrome for the first time was described in 1941 [10]. The introduction of an enzyme conjugated with an antibody and the visualization of the protein in light microscopy is due to Nakane and Pierce team [11]. The disadvantage of the IHC direct method is its low sensitivity.

Afterwards, an *IHC indirect method* was developed as follows: antigen-primary antibody, nonconjugated-secondary antibody (anti-primary antibody), conjugated with a fluorochrome or an enzyme, which convert a soluble substrate into an insoluble colored substrate [12, 13]. This method allowed the visual signal to be intensified.

The need to improve more the signal amplification has led to new changes. Thus the secondary antibody has been conjugated with other substances, such as biotin molecules, which in turn form complexes with streptavidin, forming a complex with an enzyme (e.g., horseradish peroxidase) [14]. More recently, an even more sensitive method was used in which a large number of secondary antibodies and enzymes are conjugated to a polymer chain (e.g., dextran) [15].

In the IHC technique, even *an array-like reaction* can be carried out on the same tissue section by targeting several proteins by using antibodies from different species (mouse, rabbit, goat, etc.), different enzymes coupled to the secondary antibody (e.g., horseradish peroxidase and alkaline phosphatase), different chromogens (e.g., 3,3′diaminobenzidine or 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium) or fluorochromes (e.g., FITC and rhodamine) with different colors.

 Sometimes, especially when the protein of interest is low and the immunohistochemical signal is weak or with interruptions, a confirmation for protein expression by Western blotting is required. This confirmation is also required when we are not sure whether the antibody specifically binds to the protein of interest or if there is a nonspecific antibody labeling. The Western blot technique allows the identification of the protein as it is shown below.

### **3. Enzyme-linked immunosorbent assay (ELISA)**

Old traditional ELISA technique was developed in 1971 by Engvall and Perlmann [16] and Van Weemen and Schuurs [17] and continues to be nowadays widely used

as a routine diagnostic method allowing quantitation of a large variety of proteins [18]. The single-plex ELISA, the most utilized assay method performed in 96- or 384-well plates, has played a prominent role in the quantitative and qualitative identification of analytes.

 *Direct ELISA*, the simplest type of ELISA, could accurately quantify a specific molecule with high sensitivity from a wide variety of samples, and it is faster [19]. But the signal is less amplified.

*Indirect ELISA* detection is a two-step ELISA which involves a primary antibody and a labeled secondary antibody [20]. This method presents a higher sensitivity and flexibility (different primary detection antibodies can be used with a single labeled secondary antibody). The disadvantage is the occurrence of nonspecific signals.

Beside direct and indirect detection models, two other ELISA methods appeared, to avoid false positive or false negative results, with a high specificity, suitable for complex samples, with more sensitivity and flexibility: *sandwich ELISA*  (quantify antigens between the two layers of antibodies) [21] and *competitive ELISA*  (based on a competitive binding process between the original antigen in the sample and the add-in antigen, the more antigen in the sample, the less labeled antigen is retained in the well and the weaker the signal) [22].

Another ELISA method is *ELISpot assay*, widely used to evaluate an immune response, for example, in allergies or in autoimmunity [23, 24]. This technique, performed on PVDF membranes, has advantages like specificity, sensitivity, and wide range of detection.

However, the use of ELISA for assessing multiple analytes might be time consuming due to the large number of workflows occurring simultaneously. Moreover, ELISA is designed as a solid-state immunoassay, and the use of a planar matrix can restrict immunoassay capacity, sensitivity, and detection quality [25].

Conventional single-target assays ELISA and Western blot are suitable for biomarker validation, but could be expensive, time consuming, and sample limiting. While most of the disease conditions may arise when only one single molecule is altered, more often it is the consequence of the interaction between several molecules within the inflammation milieu; therefore, studying the diseases necessitates a comprehensive perspective.

 *ELISA on a chip*. In order to improve the method, in terms of using smaller quantities of samples, shortening the reaction time, avoiding sophisticated reading equipment, and reducing costs, a group of researchers tried to miniaturize the ELIZA platform [26]. They developed an ELIZA lab-on-a-chip system (ELIZA-LOC), which allows the use of only 5 μl of sample on a miniaturized 96-well plate combined with a CCD camera [27]. This system combines three functional elements: (i) reagent loading fluidics, (ii) assay and detection well plate, and (iii) reagent removal fluidics. The description of LOM technology (laminated object manufacturing) to obtain this system using polymer sheets was made by Rasooly et al. [28].

Besides miniaturization, another novelty is the washing step that is integrated directly in ELISA plate. The authors state that using this technology, there is no need for a specialized laboratory to perform the ELIZA test.

#### **4. Western blot**

The Western blot (WB), also known as immunoblot, is an analytical and quantitative technique for identifying specific proteins in many biological samples, liquid or tissue/cellular homogenates [29]. The WB technique brings concrete and useful information that cannot be offered by other immunoassay methods. If the target protein, present in the sample, is altered qualitatively or quantitatively,

#### *Immunoassay Techniques Highlighting Biomarkers in Immunogenetic Diseases DOI: http://dx.doi.org/10.5772/intechopen.75951*

 the band thickness is changed compared to a control being downregulated or overexpressed. The WB results can also guide us to a genetic investigation in case of partial deletion or duplication in the protein gene [30]. In addition, the WB method allows a quick comparison of target protein expression in many patients in medical diagnosis.

 The WB technique was invented by Harry Towbin and co-workers in 1979. They used the method to identify bacterial or chicken ribosomal proteins separated on polyacrylamide gels containing urea. They called this method "electrophoretic blotting technique" [31]. The WB name was given 2 years later by Neal Burnette, which also brought some improvements to this method, including the use of SDS-PAGE gels [32]. The name "Western" was inspired by the earlier name of other blotting methods, "Southern", named after the name of Edwin Southern, who published in 1975 a method for detecting specific DNA sequences [33], and "Northern" whose name was inspired by the name of the first blotting technique, "Southern", a RNA detection technique, developed in 1977 by Alwine et al. [34].

Over time, the method has improved and has become easier to achieve, with nearly all materials commercially available: transfer devices, antibodies, pre-casting gels, digital imaging devices, and so on. However, in the most part, as methodology, the technique proposed by the Towbin team remains valid after 38 years. Burnette, Stark, and Towbin said after many years that they were surprised by the success and longevity of the method [35].

In summary, the Western blot method is a way to identify a target protein from a biological sample, a mixture of proteins, running it on polyacrylamide gel. The proteins in the sample are separated by SDS-PAGE gel electrophoresis, depending on their molecular weight. Because the gel is hard to handle, being fragile, the proteins are transferred to a membrane, usually nitrocellulose or PVDF (polyvinylidene fluoride), that maintains the gel pattern as a copy [7]. The electrical current causes the transfer. For visualization of the protein of interest, the membrane is probed by a specific primary antibody, it binds the specific epitope of the protein, and it is labeled by addition of a secondary antibody recognizing the primary antibody conjugated with a detection reagent (fluorophore, enzyme, and radioisotope). The visualization is done colorimetric, by chemiluminescence, on X-ray film, or directly in the membrane with the aid of an imaging system.

 In order to be able to reuse a WB membrane that has already been exposed to primary and secondary antibodies, it is necessary to wash it. This operation is called stripping. Only membranes that have been treated with ECL (enhanced *chemiluminescence kit*) for protein visualization by chemiluminescence can be reused. This method is useful when we want to investigate more proteins on the same blot, for example, a protein of interest and a loading control protein. It saves biological samples, time, and substances. For stripping, special buffers are using that can efficiently remove antibodies but do not remove too much amount of the proteins on the membrane.

The WB system size may vary, with electrophoresis/transfer tank, gels, and membrane: mini, midi, and large, depending on the investigated protein size and the time needed for separation. However, the vast majority of investigators use now the mini system, sometimes the midi one, because of the existence of gradient gels and more sophisticated devices (see below). Transfer systems were developed by few companies to allow proteins the migration from gel to the membrane in different ways, using varying amounts of buffers: wet, semidry, or dry systems. New digital technologies offer a good and rapid bands visualization, avoiding underexposure or overexposure, as in the case of X-ray film developing. The images can be stored in a computer database and can be analyzed with software that measures the optical density of the bands.

#### **4.1 Other methods based on the Western blotting technique**

#### *4.1.1 Multiplex Western blot*

 In the last few years, it has become a necessity to analyze multiple target proteins at the same time, in order to compare the expression of proteins involved in a specific pathology. First Multiplex WB experiment (multiplex Western blot (MWB)) was optimized by Anderson and Davison [36] to study different muscle proteins involved in muscular dystrophies. This method allowed a simultaneously screening of multiple proteins with a different size on a pair of blots, using a cocktail of monoclonal antibodies which permitted the identification of primary deficient and second deficient proteins in several muscle pathologies, knowing that the primary reduction of a protein causes the secondary reduction of another protein. The use of a MWB allowed establishing a biomarker profile for each patient, providing valuable information for diagnosis as well as for phenotype-genotype correlations. The MWB method proposed by Anderson used a biphasic polyacrylamide gel (with different concentrations) system electrophoresis, which separated the proteins with different molecular weights: molecular mass more than 200 kDa in the upper part of the gel, with 5.5–4% polyacrylamide gradient, while proteins with molecular mass under 150 kDa are separated in the lower phase, 7% polyacrylamide gel.

Introduction of this technique has revolutionized the medical diagnosis and opened new perspectives in biomedical research. Simultaneous analysis of several proteins involved in different pathologies by MWB reduced the cost and time for analysis. By this method, it could be determined and compared proteins in the same sample as well as a secondary reduction of other proteins in a specific disease [37, 38].

#### *4.1.2 Capillary electrophoresis and capillary Western blotting*

By this method, the molecules are separated by the size inside a capillary filled with an electrolyte. The advantage of the method is that the separating sieve matrix can be automatically pumped in and out because it contains rather unknown polymers than the typical cross-linked polymers for the gels. The big difference between the classical method and this one is that many samples can be run over and over again in an automated manner that saves a lot of time [35].

Capillary electrophoresis (CE) needs a smaller amount of sample than SDS-PAGE and offers a better resolution of a protein size. Proteins travel down the capillaries and are spaced according to the size. When the individual proteins reach the end of the capillary, they drop on a blotting membrane that moves along the capillary opening. It has been shown that classical protein standards such as carbonic anhydrase and lysozyme can be separated within an hour using only a few nanoliters of the sample [39].

 O'Neill et al. [40] have been able to capture the resolved proteins on the capillary wall by photochemically activated molecules. This method allowed immune complexes to be formed after electrophoresis, in the capillary. Chemiluminescence reagents flowed through the capillary, and the image was taken with a CCD camera [40].

#### *4.1.3 Microfluidic Western blotting*

This technology reduces much more the amount of the sample required for WB and also the length of the capillaries from centimeters to microns, using microfluidic channels. He and Herr, in 2009, developed an automated immunoblotting method using a single streamlined microfluidic assay. A glass microfluidic chip, which has integrated a PAGE electrophoresis with subsequent in situ

#### *Immunoassay Techniques Highlighting Biomarkers in Immunogenetic Diseases DOI: http://dx.doi.org/10.5772/intechopen.75951*

 immunoblotting, allowed a rapid protein separation, directed electrophoretic transfer of resolved proteins to an in-line blotting membrane, and a high-efficiency identification of proteins of interest using antibody-functionalized membranes [41, 42]. The system requires only 0.01–0.5 μg of protein.

#### *4.1.4 Dot blot*

It is a method very similar to WB, but the proteins are not separated by gel electrophoresis. The samples are applied in small dots directly on the membrane and then spotted through circular templates. After membrane drying, the antibodies are applied. The visualization of target protein is made like at WB, chemiluminescent or colorimetric. Dot blot is used to test the specificity of some antibodies, to test the antibody concentration used for WB, or to evaluate the presence of a target protein in the sample before WB.

#### *4.1.5 Far-Western blotting*

It is used to detect a protein-protein interaction *in vitro*. Instead of the primary antibody for detecting the protein of interest, this method uses a nonantibody protein that binds to the protein of interest. Far-Western blotting detects proteins on the basis of the presence or the absence of binding sites for the protein probe. This method is important in characterization of protein interactions in biological processes such as signal transductions [43], receptor-ligand interactions, or screen libraries for interacting proteins.

#### **5. Protein microarray**

Protein microarray analysis has an increasingly use both for research purposes as well as for various biomedical applications, *including the niches ones* like evaluating markers of apoptosis activated by various therapies such as photodynamic therapy (PDT), assessment of epigenetic milieu, or transcriptional activity in treated cells [44]. Thus, protein microarray is a proteomic tool that can deliver high-throughput data for revealing new therapeutic targets [45].

 Protein microarray history has spanned the last two decades, the basic principle being identical with ELISA, but there are several advantages such as spotting in terms of miniaturization, multiplexing, and large data obtained in an ELISA equivalent time. Briefly, biological samples of interest (e.g., serum, plasma, etc.) are incubated on a slide containing immobilized antibodies, proteins, or peptides. An antigen-antibody reaction occurs between an analyte from the tested sample and the corresponding antibody from slide followed by the detection step through various methods (e.g., fluorescence-based detection). The slide is further scanned, followed by image acquisition, data processing, and analysis. There are several classifications of this technique, but it could fall into two main categories: direct phase (e.g., antibody-, protein-, peptide array) and reverse or indirect phase where sample of interest is spotted on a slide and the corresponding antibody is further added.

Among all these variants, the antibody array type is preferred in tumor research domain or in biomarkers discovery/quantification due to technique's high versatility and reproducibility [46]. The reverse phase array format could also be used for biomarker discovery because it is specificity but has the disadvantage of being more laborious.

It is worth to emphasize that protein microarray could be customized in terms of number and multiplicity of tested analytes one achieving new research and clinical

benefits through this technology. Thus, although fundamental research purposes prevail when it comes to array platforms, there is also a recent increasing trend in clinical research, diagnostics, and even industry applications such as pharmacy or food. For instance, recent attempts are made in using array platform for autoimmune disease insights. Thus, novel antigen arrays have been developed in order to discover new autoantibody targets, providing analysis for hundreds of samples and of their reactivity pattern against thousands of antigens simultaneously [47].

 Customizing an array in relation to clinical purpose confirms the flexibility of these platforms in assisting molecular management of the disease. A customized platform was designed in order to monitor severe acute respiratory syndrome (SARS) infection by screening hundreds of sera based on the reactivity against certain selected proteins from SARS coronavirus. Authors have reported that with this customized array, viral infection could be monitored for many months after infection [48]. This type of microarray platform has been further updated to a serological assay for the specific detection of IgM and IgG antibodies against the S1 receptor-binding subunit of the spike protein of emerging human coronavirus hCoV-EMC and SARS-CoV as antigens [49].

 Protein microarray is a technology in continuous evolution offering multiple possibilities in updating other proteomic techniques. Therefore, the development of the "microwestern array" is a clear proof how traditional methods like Western blot can be linked to novel technology, thus significantly expanding the research technological arsenal [50].

Data generated by ELISA and WB require sometimes additional complementary proteomic methods to supplement and even support the scientific information. Such supportive task is often accomplished by protein microarrays providing important evidence on modulation of signaling networks and potential targets (or pathways); these factors or networks must first be identified, and array platforms allow exactly this development by exploring dozens of targets simultaneously within a single sample, providing lots of data which may be further investigated using traditional ELISA or WB techniques [51].

#### **6. Luminex xMAP array**

 An important improvement in the biological assay field was made in the late 1990s when Luminex xMAP technology was launched. xMAP technology combines the principles of ELISA and flow cytometry, but goes beyond the limitations of solid-phase reaction kinetics and is suitable for high throughput, multiplex, and simultaneous detection of different biomarkers within a very small volume sample. Bringing together advanced fluidics, optics, and digital signal processing with proprietary microspheres, xMAP technology became one of the fastest growing multiplex technologies. Featuring a flexible open-architecture design, xMAP technology enables the configuration of various assays, quickly, cost effectively, and accurately, useful in clinical and research laboratories [52].

A key component of Luminex xMAP technology is represented by proprietary color-code polystyrene microspheres (beads) internally dyed with precise concentrations of two or three spectrally distinct fluorochromes. Through precise concentrations of these specific dyes, up to 500 distinctly bead sets can be developed, with a different spectral signature.

Based on fluorescent reporter signals, high-speed digital-signal processors identify each individual microsphere and quantify the result of every bioassay. The capability of adding multiple conjugated beads to each sample results in the ability to obtain multiple results from each sample [53].

*Immunoassay Techniques Highlighting Biomarkers in Immunogenetic Diseases DOI: http://dx.doi.org/10.5772/intechopen.75951* 

There are different types of advanced detection platforms (as depicted in **Table 1**), and therefore, various biomarker panels could be analyzed. Accordingly, validation of novel biomarkers into multiplex immunoassay panels confers an attractive prospect of simultaneous measurement of multiple analytes in a single patient sample, enabling progres-sion monitoring and outcome prediction, even detecting major diseases in its earliest stages [54].



#### **Table 1.**

*Advantages and disadvantages of immunoassay methods presented in this chapter: IHC, ELISA, Western blot, bead-based array—Luminex technology, and chip-based array—protein microarray, SPRi.* 

Some of our studies illustrate significant dysregulation in circulating levels of cytokines and angiogenic factors in brain tumors, with over threefold upregulation of IL-6, IL-1 beta, TNF-alpha, and IL-10 and up to twofold upregulation of VEGF, FGF-2, IL-8, IL-2, and GM-CSF, with implications in tumor progression and aggressiveness, and also involved in disease-associated pain [55, 56].

 Currently, Western blot is used to validate/confirm the identified biomarkers, and association between the xMAP technology and the Western blot was remarked in many studies. Interestingly, one of them emphasized the improvement in diagnostic sensitivity of HIV infection in early stages using xMAP technology, increasing the chances of an early accurate diagnosis. Thus, it was observed a superior sensitivity of Luminex xMAP compared with Western blot. Out of 87 confirmed HIV positive cases, Western blot confirmed 74.7% sensitivity, while Luminex xMAP identified 82.8% sensitivity (p < 0.05) [57]. Further advancements will be needed for a successful validation of current discoveries, and sustained efforts are necessary to expand the translation into clinical applications toward personalized medicine [58].

#### **7. Surface plasmon resonance imaging, lab-on-a-chip**

Since our goal is not describing surface plasmon resonance imaging (SPRi) methodology, we will not insist very much on the description of the technique. We will make a brief description of the principle on which SPRi is based.

 The first SPR immunoassay was proposed by the team Liedberg, Nylander, and Lundström, in 1983 [59]. The SPR immunoassay method is label-free (unlike ELISA); no label molecule is required for analyte detection [60]. Moreover, the measurement is done in real time, which allows monitoring of the individual steps of this technology. SPRi is currently one of the most sensible platforms for studying a wide variety of interaction affinities [61, 62], involving nucleic acid sequences [63–65], peptides [66], proteins [67, 68], and carbohydrates [69]. It is possible to monitor hundreds of molecular interactions simultaneously.

The composition of a biochip consists essentially of a glass prism, coated with a thin gold film and a pre-functionalized surface chemistry. The sample to be

#### *Immunoassay Techniques Highlighting Biomarkers in Immunogenetic Diseases DOI: http://dx.doi.org/10.5772/intechopen.75951*

analyzed is injected over the biochip, and the detection of a specific molecule can be performed by immobilizing a binding partner on the biochip. SPRi makes a nonlabeling and a real-time detection of biomarkers [70].

The SPRi platform allows the quantitative detection of multiple simultaneous multiplex interactions, and many studies are based on this application for screening a variety of analytes in different array types. The main advantage of using SPRi in immunodiagnostics is the possibility of monitoring the antigen-antibody reaction in real-time, estimating kinetics, how quickly it occurs and how durable it is. In addition, it does not require any labels.

 In comparison to ELISA and Western Blot, SPRi has the advantage of investigating a large number of different analytes from the same sample (several hundred different spots can be placed on a biochip), and after washing the biochip, it is possible to immediately analyze another sample. SPRi takes less time than other methods. The disadvantage of SPRi would be that only liquid biological samples (blood, urine, cerebrospinal fluid, and cell culture medium) can be analyzed, and it does not analyze biological samples from different tissues/tissue lysates. SPRi is very effective when there are many samples and many different interactions to analyze, but for a small number of samples or to demonstrate only one type of interaction between two proteins, for example, WB is more efficient.

As a conclusion regarding the technologies presented in this chapter, we show **Table 1** with the advantages and disadvantages of each method.

### **8. Immunoassay methods in immunogenetic disease diagnostic**

*Indirect immunofluorescence (IIF)* for autoantibody analysis is one of the routine diagnostic methods. Different tissue sections or human tumor cell lines—HEp-2 are used as the source of antigen over which the serum of patients with specific autoantibodies is applied [71].

The antinuclear antibody (ANA) test is such a standard screening assay. The American College of Rheumatology declared HEp-2 IIF as the preferred method for ANA screening [72]. The large amount of ANAs can indicate an autoimmune disease, including systemic lupus erythematosus, Sjogren's syndrome, scleroderma, rheumatoid arthritis, idiopathic inflammatory myopathy, and others.

One of the immunohistochemistry method applications in autoimmune disease diagnosis is the detection of the presence of MHC I and, more recently, of MHC II in skeletal muscle of patients with idiopathic inflammatory myopathies (IIMs). It is a group of autoimmune systemic diseases, of which the most common forms are dermatomyositis, polymyositis, and inclusion body myositis. The study of muscle biopsy makes the difference in diagnosis between subtypes, but also among other types of myopathies and IIMs. In addition to other pathological features, the presence of MHC I and MHC II in sarcolemma gives the certainty of diagnosis, as long as they are not present in normal muscles [73–76]. Their overexpression in IIMs is induced by cytokines, including interferon and tumor necrosis factor alpha (TNF alpha) [77]. A study of 120 muscle biopsies from patients with different forms of IIMs showed a presence of MHC I in all biopsies and MHC II in 93% of them [76].

The MHC I expression appears early and precedes the lymphocyte infiltrate [78], persisting in late disease, and it is not attenuated by immunosuppressive treatment [79–81].

MHC II expression on antigen presenting cells activates T-helper cells and initiates an immune response without knowing the mechanism by which MHC II alleles mediate susceptibility to a given autoimmune disease [82].

From our experience in IIM cases where the IHC is not conclusive, a WB verification or validation is of great help in highlighting MHC I and II bands at their specific molecular weight.

From the more recent studies, we mention that the anti-signal recognition particle antibodies in the serum of IIM patients have diagnostic and prognostic value especially in the forms of immune-mediated necrotizing myopathy [83]. The authors draw attention to a mandatory IIF test along with the dot immunoassay to avoid false positive results from the latter method in pathologies not associated with IIM. The results sometimes depend on the nature of the antigen used in the technique and can be denatured.

 *ELISA* is used as a diagnostic tool in autoimmune diseases, for evaluation of serum autoantibodies. Antinuclear antibodies (ANAs) directed against a variety of nuclear and cytoplasmic antigens are found with a high frequency in many systemic autoimmune disorders like systemic lupus erythematosus, scleroderma, Sjogren's syndrome, myositis, etc. *ANA-HEp-2 Screen ELISA* is an immunoassay method for the qualitative combined detection of IgG antibodies against human serum HEp-2 cells. Each well is coated with Hep-2 cellular lysate. The test detects in a well plate total ANAs against double stranded DNA, histone, SS-A (Ro), SS-B (La), Sm, snRNP/Sm, Scl-70, PM-Scl, Jo-1, and centromeric antigens.

 HEp-2 cells allow the recognition of over 30 nuclear and cytoplasmic patterns that are given upwards of 50 different autoantibodies [84, 85]. The specificity of the test is closely related to the quality of the antigens used [86]. It is one of the most common methods of diagnosis in organ-specific autoimmune diseases, such as Grave's disease, primary biliary cirrhosis, insulin-dependent diabetes mellitus or systemic autoimmune disorders affecting different organs, such as systemic sclerosis, Sjogren's syndrome, and mixed connective tissue disease rheumatoid arthritis [87, 88].

From recent research studies [89], we want to mention cortactin antibodies as new biomarkers in double seronegative myasthenia gravis (myasthenia gravis form dSNMG). ELISA tests validated by WB have demonstrated that the presence of cortactin autoantibodies is a biomarker to be taken into account, suggesting that the disease will be ocular or mild generalized and could be done routinely in the future.

Another work on rheumatoid arthritis shows that, apart from the autoantibody system that recognizes citrullinated proteins, the identification of another antibody system against carbamylated proteins has an important early diagnostic value, predicting a more severe course of disease [90]. The ELISA method used in this study could become routine for serum testing of patients with rheumatoid arthritis.

*Western blot*. To highlight the importance of WB technique in clinical diagnosis, we give some eloquent examples below. The WB method has been used in many studies, along with immunoprecipitation, ELISA, and flow cytometry, to demonstrate the quantitative or qualitative modification of proteins of interest in autoimmune diseases in order to find new biomarkers or therapeutic targets. WB has proven to be a good tool for serological tests.

*Line-blot immunoassay* is a Western blotting method that uses recombinant antigens immobilized on straight lines on a nylon strip. They are incubated with patient serum containing autoantibodies. They bind to the antigens present on the strip and are viewed colorimetrically. Interpretation of the results is done by comparing the color intensity of strips with the color of strips of a positive standard. There are some studies that have shown the utility of this method in the detection of autoantibodies present in serum but which could not be identified by IIF, for example, anti-SS-A/Ro in Sjogren's syndrome [91, 92].

Haroon et al. have demonstrated using the WB method that there is an interaction between endoplasmic reticulum aminopeptidase 1 (ERAP1) with human

#### *Immunoassay Techniques Highlighting Biomarkers in Immunogenetic Diseases DOI: http://dx.doi.org/10.5772/intechopen.75951*

leucocyte antigen (HLA)-B \*27 in ankylosing spondylitis [93], and that the HLA-B27 molecules could alter the ERAP1 level. The functional interaction between ERAP1-peptide and HLA-B27 could thus be the missing link in the pathogenesis of ankylosing spondylitis.

Stagakis et al. studied whether anti-TNF therapy improves insulin resistance in rheumatoid arthritis [94]. Western blot was used to analyze the proteins p-Ser312 IRS-1 and p-AKT from peripheral blood mononuclear cell lysates. It has been established that anti-TNF alpha therapy has a positive effect, improving insulin sensitivity and reversing the defects in signaling insulin cascade in this disease.

Tsui et al. have conducted a study of the serum levels of noggin (NOG) and sclerostin (SOST) in patients with ankylosing spondylitis, more specifically, on the immune response to these two molecules [95]. The WB method was used to quantify the relative amounts of NOG/SOST-IgG immune complexes. An increased level of NOG/SOST-IgG immune complexes was found in patients with this pathology.

 Rizzo et al. [96] showed that the dimeric form of the HLA-G molecule is associated with the response to methotrexate treatment in patients with early rheumatoid arthritis. HLA-G dimeric and monomeric forms have been highlighted by WB. The presence of dimeric form in plasma prior to methotrexate therapy could be a biomarker for the patient's response to treatment.

 *Protein microarray*. Antibody microarrays could provide a real-time vision of biological processes, such proteomic instrument being used in clinic to analyze serum and plasma in several pathologies including autoimmune disorders. One of the autoimmune diseases approached through protein array is *systemic lupus erythematosus* (SLE) where SLE clinical heterogeneity and the absence of robust biomarkers to evaluate the disease states and differentiate from other autoimmune conditions are yet to be resolved [97]. Thus, using an antibody-based leukocytecapture microarray, mononuclear cells isolated from peripheral blood of 60 SLE patients were processed for obtaining proteomic patterns to distinguish SLE from healthy subjects. With this array platform, it was improved the conventional SLE diagnostics and disease states elucidation [98]. Moreover, an "in-house" antibody microarray comprising 135 human recombinant single-chain fragment variables aiming various immune proteins were used to examine *systemic sclerosis* (SSc) and SLE patients. This tailored array identified a significant number of differentially expressed proteins that delineate SLE from systemic sclerosis, thus surpassing disease classification through conventional clinical parameters, including, ANA, anti-DNA, SLEDAI-2 k, C1q, C3, C4, and CRP [99]**.**

Another challenging field for protein array is related to *rheumatoid arthritis* (RA) as it could detect biomarkers specific for arthritis and not for autoimmune diseases in general [100]. Some research groups have started to develop different antigen arrays for differential diagnosis and even RA molecular classifying. Panels of proteins were detected, among these three proteins, namely, WIBG, GABARALP2, and ZNF706, were suggested as potential specific markers for RA early stages [101]**.** Hence, protein arrays bring valuable data to immune-disease background allowing exploration of numerous samples in parallel and thousands of targets.

Antibodies against ion channels, receptors, synaptic proteins, etc. confirm protein microarrays as a future potential tool in routine diagnosis [102]**.** Whatever commercially available or customized platforms, antibody arrays start to emerge in clinic by designing *omics* disease signatures helping the disease management**.**

The protein microarray was used in a study of *pemphigus vulgaris*, an autoimmune skin disease, to identify the entire set of antibodies, bringing extra data about the complex relationship between genetics and disease development [103]. The results were correlated with those obtained by the ELISA and proved to be compatible. The main targets for autoantibodies are desmoglein-3 and 1, but the

study showed that there are autoantibodies that are not directed to desmoglein at a significant number of patients.

A study regarding *ankylosing spondylitis* using the protein microarray, confirmed by ELISA, to characterize anti-ankylosing spondylitis autoantibodies, showed that anti-protein phosphatase 1A (PPM1A) autoantibodies are present in the serum of the patients and that they could serve not only as biomarkers for diagnosis, disease severity, and response to anti-TNF therapy, but also as a therapeutic target [104].

*Luminex xMAP technology* has developed as an alternative to planar microarray methods. Bead-based immunoassays are one of them. The determinations by this method and by ELISA of anti-thyroid peroxidase and anti-thyroglobulin antibodies in autoimmune diseases have been shown to be compatibles [105].

There is a commercially available kit for ANA detection, which is low cost and saves time. Antigens corresponding to autoantibodies are linked to polystyrene microspheres labeled internally with different amounts of two different fluorochromes, resulting in 100 different color spectra. Each microbead carries an antigen specific for a single antibody [106].

Good results were obtained in assessing a number of antinuclear autoantibodies as: dsDNA, Sm and Sm/RNP (in systemic lupus erythematosus), SS-A/Ro and SS-B/La (in Sjogren's syndrome), Jo-1 (myositis), ribosome (systemic lupus erythematosus), and centromere (systemic sclerosis) [107].

One of the problems with this technology could be the lack of a true quantitative calibration due to the difference in affinity of the antibodies for the antigen [108]. Some authors argue that it is also necessary to validate the results by other immunoassay methods [106], while others claim that the accuracy of the technique is similar to that observed by ELISA [109, 110].

There are studies in which the Luminex methodology is used for the analysis of serum biomarkers in various autoimmune diseases. Thus, in an article on ankylosing spondylitis, certain cytokines as hepatocyte growth factor (HGF), CXCL8, and matrix metalloproteinases (MMP-8 and MMP-9) identified from a large number of biomarkers by Luminex could be diagnostic targets, their serum levels being increased in this disease [111].

 In other chapter regarding ankylosing spondylitis, Luminex bead-based technology was used for serum cytokines analysis, and the conclusion was that the utilization of TNF alpha inhibitors decreases the number of T cells producing proinflammatory cytokines [112].

Mou et al. showed, using Luminex technology in combination with PCR, that in ankylosing spondylitis patients from Southern China with HLA-B27 in their serum, HLA-B2704 subtype predominates. And the HLA-B2715 subtype may have a disease prognostic value, early onset being related to this subtype [113].

*Surface plasmon resonance imaging*. Despite its great sensitivity, this technology is relatively little used to determine the concentration of some analytes. Improving signal amplification methods is one of the research goals in this technique.

In some autoimmune diseases, such as rheumatoid arthritis (RA), psoriatic arthritis, systemic lupus erythematosus, or Sjogren's syndrome, autoantibodies attack citrullinated proteins, and the presence of anti-citrullinated proteins, antibodies is a standard test in these cases. The use of SPRi for monitoring autoantibodies that bind to different citrullinated targets was first described by Lokate et al. SPRi has shown its ability to detect the interaction between citrullinated peptides and serum autoantibodies in RA patients in one step [114].

 SPRi microarray technique was also used in a more recent research to identify autoantibodies against citrullinated protein (ACPA) profiles in patients with early onset rheumatoid arthritis. The authors made a comparative study using citrullinated

#### *Immunoassay Techniques Highlighting Biomarkers in Immunogenetic Diseases DOI: http://dx.doi.org/10.5772/intechopen.75951*

and noncitrullinated peptides [115]. The study showed that SPRi is a suitable methodology for detecting ACPAs in the serum of patients with this pathology.

A subsequent study was also revealed by SPRi, the presence of citrullinated B-cell epitopes in fibrinogen [116].

A research team [117] showed the use of SPRi and gold particles to amplify the signal for the detection of inflammation biomarker TNF-alpha in serum. Also, the use of a specific buffer solution for sample dilution was utilized to reduce the nonspecific binding in real samples. Thus, a low limit of detection, as well as a good reproducibility and the longevity of chips, is a good motivation to use this immunoassay method to detect biomarkers that are in low concentrations in biological samples.

Buhl et al. reported in a research paper the use of SPRi technology for the antidsDNA detection in systemic lupus erythematosus [118].

#### **9. Conclusions**

Immunoassay methods have many advantages but some limitations too. Their importance in identifying different biomarkers for diagnosis or personalized therapy is essential. That is why they have diversified so much, in order to be able to answer all the challenges. Additionally, these methods and technologies have also specialized in an advanced degree, so that they can detect smaller amounts of molecules with as high a precision as possible in a shorter time. The antigenantibody response gives them great sensitivity. The development of more advanced equipment leads to the automation of these methods and to a greater efficiency, with applicability in diagnosis and therapeutic monitoring, in discovery of new biomarkers and even in pharmacology.

In order to be used for diagnosis in different laboratories, these methods and kits should be standardized. The problems to be posed are: the clinical manifestation of the disease in different individuals, the source of the antigen, the specificity and sensitivity of the autoantibodies for different antigens, the reproducibility of the assay, and the precision and the accuracy of the method [91, 119, 120].

Some studies show a good correlation between IIF and ELISA methods [84, 121, 122], and others, on the contrary, show different results between these methods [71, 123].

 Multiplex technologies are gaining more and more followers in recent years by allowing simultaneous analysis of a multitude of analysts, saving time and costs. However, there are studies showing that compared to the old methods, some false negative or false positive results are obtained [124–128]. Cross reactivities may also occur [129].

Assay kits produced by different manufacturers can show variable results also. More than this, the methodology used by each laboratory can lead to different results, even by using the same kit. International standardization is required. A collaboration between an international body and organizations responsible for quality of assessment of assays is desirable, so that a collaboration among clinicians, diagnostic laboratories, and manufacturers to be established.

#### **Acknowledgements**

All authors contributed equally to this work. Partially supported from Project ID: SMIS CSNR 1882/49159 (CAMED), PN-III-P1-1.2-PCCDI-2017-0341 (PATHDERM), PN-III-P1-1.2-PCCDI-2017-0782 (REGMED), PN 16.22.02.04/2016, PN 16.22.02.05/2016, PN 18.21.02.02/2018, and PN 18.21.01.06/2018.

## **Author details**

Emilia Manole1,2\*, Alexandra E. Bastian3,4, Ionela D. Popescu1 , Carolina Constantin1,2,5, Simona Mihai1 , Gisela F. Gaina1,5, Elena Codrici1 and Monica T. Neagu1,2,5

1 National Institute of Pathology "Victor Babes", Bucharest, Romania

2 Colentina Clinical Hospital, Research Centre, Bucharest, Romania

3 Pathology Department, Colentina Clinical Hospital, Bucharest, Romania

4 University of Medicine "Carol Davila", Bucharest, Romania

5 Faculty of Biology, University of Bucharest, Bucharest, Romania

\*Address all correspondence to: emilia\_manole@yahoo.com

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Immunoassay Techniques Highlighting Biomarkers in Immunogenetic Diseases DOI: http://dx.doi.org/10.5772/intechopen.75951* 

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**73**

tuberculosis.

**1. Introduction**

Th1-type cytokines, IL17, iNOS3

**Chapter 5**

**Abstract**

Immunogenetic and

*Gloria Guillermina Guerrero Manriquez*

Immunotherapy in Tuberculosis

Tuberculosis (TB) is an infectious disease caused by *Mycobacterium tuberculosis* (MTb). TB causes mortality of millions of people every year. *Mycobacterium bovis Bacillus Calmette Güerin* (BCG) is the only officially approved vaccine that protects against miliary TB and children but fails to protect in adulthood presumably because of the lack of long lasting immunological memory. The problem is even more aggravated because of the emergence of multidrug-resistant strains. Therefore, immunogenetics and immunotherapy of antimycobacterial immunity are complex and poorly characterized. However, several studies either in the mouse model or *in vitro*, using derived dendritic or macrophages derived from PBMCs or human cell lines, have shown that Th1 type cellular immune response represented by IFN-γ, IL-12 in conjunction with IL-17, and IL-23 are key players of the immune protection to M. *tuberculosis*. It is known that under different settings type I IFNs promote bacterial virulence and disease exacerbation, since a study with active TB patients was concomitant with a dominant neutrophil-driven interferon inducible gene pattern. Furthermore, in an independent cohort of TB patients, ex vivo experiments with BMDCs (bone marrow–derived dendritic cells) and myeloid from lung showed that there is a cross action between the components of IL-1β, eicosanoid pathways (prostaglandin, lipoxins, and leukotrienes) in active TB, while excessive type I IFNs and IL10 induction, concomitant with an inhibition of iNO3 and prostaglandin, could be found. These responses could be used as a therapeutic target instead of any other treatment based on antibiotics. Furthermore, the work from us has demonstrated that interferon alpha plus BCG vaccine protects against mycobacterial infections through modulating the Th1-type cellular immune response, iNOs, and IL-1β production. These immunomodulatory properties of interferon alpha could influence the outcome of the innate and acquired host immune responses in

**Keywords:** type I IFNs, adjuvants, *mycobacterial* infections, BCG vaccine,

Tuberculosis is the most serious cause of mortality after HIV/AIDS [1, 2]. Until now, BCG is the only officially approved vaccine that protects against miliary TB in children but it fails to protect in adulthood [1–3]. Therefore, the search for subunits agents that can boost primarily the central memory is still an issue of intense research worldwide [1, 2]. Several candidates have been developed and are under clinical studies [4–8]. Type I IFNs emerge, thus, as a

## **Chapter 5**

## Immunogenetic and Immunotherapy in Tuberculosis

*Gloria Guillermina Guerrero Manriquez* 

## **Abstract**

 Tuberculosis (TB) is an infectious disease caused by *Mycobacterium tuberculosis* (MTb). TB causes mortality of millions of people every year. *Mycobacterium bovis Bacillus Calmette Güerin* (BCG) is the only officially approved vaccine that protects against miliary TB and children but fails to protect in adulthood presumably because of the lack of long lasting immunological memory. The problem is even more aggravated because of the emergence of multidrug-resistant strains. Therefore, immunogenetics and immunotherapy of antimycobacterial immunity are complex and poorly characterized. However, several studies either in the mouse model or *in vitro*, using derived dendritic or macrophages derived from PBMCs or human cell lines, have shown that Th1 type cellular immune response represented by IFN-γ, IL-12 in conjunction with IL-17, and IL-23 are key players of the immune protection to M. *tuberculosis*. It is known that under different settings type I IFNs promote bacterial virulence and disease exacerbation, since a study with active TB patients was concomitant with a dominant neutrophil-driven interferon inducible gene pattern. Furthermore, in an independent cohort of TB patients, ex vivo experiments with BMDCs (bone marrow–derived dendritic cells) and myeloid from lung showed that there is a cross action between the components of IL-1β, eicosanoid pathways (prostaglandin, lipoxins, and leukotrienes) in active TB, while excessive type I IFNs and IL10 induction, concomitant with an inhibition of iNO3 and prostaglandin, could be found. These responses could be used as a therapeutic target instead of any other treatment based on antibiotics. Furthermore, the work from us has demonstrated that interferon alpha plus BCG vaccine protects against mycobacterial infections through modulating the Th1-type cellular immune response, iNOs, and IL-1β production. These immunomodulatory properties of interferon alpha could influence the outcome of the innate and acquired host immune responses in tuberculosis.

**Keywords:** type I IFNs, adjuvants, *mycobacterial* infections, BCG vaccine, Th1-type cytokines, IL17, iNOS3

#### **1. Introduction**

Tuberculosis is the most serious cause of mortality after HIV/AIDS [1, 2]. Until now, BCG is the only officially approved vaccine that protects against miliary TB in children but it fails to protect in adulthood [1–3]. Therefore, the search for subunits agents that can boost primarily the central memory is still an issue of intense research worldwide [1, 2]. Several candidates have been developed and are under clinical studies [4–8]. Type I IFNs emerge, thus, as a

potential candidate adjuvant in bacterial infections. More than half century ago, interferons were first described like an antiviral "activity" [9–12]. Later on, they were recognized as innate inflammatory cytokines, and considered to be major connector of the innate and adaptive immunity. In general, type I IFNs could be considered like pleiotropic cytokines that belong to a multigenic family as outlined in **Table 1** [11, 12].

 Plasmacytoid dendritic cells (pDCs) are known to be major producers of type I IFNs producing up to hundred to a thousand times more IFNs-α than other cell types [13, 14]. To be produced, a recognition between pathogen-associated molecular patterns (PAMPs) on the pathogen surface (viral and bacterial), Tolllike receptors (TLRs) (bacterial), with the pattern recognition receptor (PRRs), antigen-presenting cells (dendritic cells and macrophages) is necessary; followed by the activation of Myd88, interferon regulatory factor 3 (IRF3), IRF5 and IRF7 (IFN-α), and NFκβ [13, 15]. Except leucocytes (which produce primarily IFN subtypes), all cells are capable of detecting intracellular PAMPs and producing IFN-β following activation of IRF3 and NF-κβ [14, 16]. After viral or bacterial infections, there is an increase in the IFNs production in different types of cells. The functions *in vivo* of type I IFNs are the activation of DCs (dendritic cells), critical antigen-presenting cell for initiating immunity [13], in fact, type I IFN-treated DCs prime T cells *in vitro* promote the expression of costimulatory molecules [15], stimulate human blood monocytes differentiation into DCs [15]. Regardless of its role as an antiviral agent [11, 12], type I IFNs are also able to enhance adaptive immunity. A huge body of studies have shown type I IFNs immunomodulatory properties either to virus as well as to bacteria infections [12–15]. We think in agreement with other groups that type I IFNs have a strikingly dichotomy behavior, since their actions can be either positive or negative depending on the settings and the surrounding scenery that will strongly influence the outcome of the host immune response.


**Table 1.**  *The multigenic family of type I IFNs in nature.* 

## **2. The type I IFNs in nature**

 As outlined in **Table 1**, several human type I IFNs are already known to be selectively produced in a tissue-specific. As a multigenic family, type I IFNs, in particular, IFN-alpha, are comprised of 13, while IFN-β, IFN-ε (genital tract), IFN-κ (keratinocytes), and IFN-ω are only coded for a single gene. For the signalization to be carried out, there are basically two main steps that are common to the 17 IFNs. First is the binding to and signal through a shared heterodimeric receptor complex composed of a single chain of IFNAR1 and IFNAR2, which is present in almost on all nucleated cells [13–15]. Second, a signal is propagated within the cell via the JAK-STAT signaling pathway [13–15]. This is also common to type III IFNs. As occurred in other interaction receptor-ligand, there are low or high affinity binding, and this could impact in the stability and the variety of the complex formed and therefore in the outcome of the host response [13–15]. This point has been the focus of intense research, because many questions arise for this interaction. Thus, for example, it is intriguing: why some interferons signal through the same receptor? Is there a redundancy of the immune system or is tailoring for each type of pathogen? Is the molecular evolution that has an impact also in the transcriptional gene printing, or in the adjuvant activities?

One of the hallmarks of the IFN action in nature is its immunomodulatory behavior [7, 10, 17]. These include among others the role of type I IFNs in the connection of innate and adaptive immune responses, such as B activation for enhancement of Ab responses [7, 10, 18], promotion of Th1 responses in terms of IgG2a Ab production, and CD4 + T cells activation and induction of an *in vitro*  and *in vivo* differentiation of monocytes into functionally active DC [8, 19, 20], NK and T cytolytic activity, upregulation of histocompatibility antigen class I expression, induction of proliferation, and long-term survival of memory CD8 + T cells [7, 19, 20].

## **3. Is there any specificity in the type I IFN induction?**

 At glance yes, it would seem that there is specificity in the type I IFNs induction. As highlighted above, type I IFNs induction is a consequence of the hostpathogen interaction [10, 16]. Thus, while membrane-bound PRRs are endowed with the ability to recognize viral or bacterial PAMPS (located in the cell surface, and within endosomal compartments [20]), it could be possible that the expression profile of each cell type in particular of these PRRS on the innate immune cells that could potentially give rise to specificity in IFN subtype production—an early step during infection inward ultimately fine-tuning the immune response an issue that is challenging because to measure the different profiles of IFN-α for each cell type has enormous limitations under physiological conditions, but it is true that should be pinpointed whether the IFN responses are qualitatively different in response to distinct pathogens [9, 20]. Furthermore, IFN-β and/or the IFN-α subtypes signal through TLRs (TLRs are membrane-bound compartments) of cosmopolitan expression in different human cells, which can potentially give some specificity to the interaction. Thus, it is known from the literature that TLR3, TLR7, TLR8, and TLR9 recognize viral nucleic acids [9, 10, 16]. Another type of receptor, specialized in detecting pathogen-derived RNA in the cytoplasm, that is also involved in the production of IFN-β in nonimmune cells, is the members of the RIG-I-like receptors (RLRs), a family of cytoplasmic RNA helicases important for host viral responses and includes retinoic acid-inducible

#### *Immunogenetics*

gene I (RIG-I)-melanoma differentiation-associated protein 5 (MDA5) and the laboratory of genetics and physiology-2-(LGP3). The signalization through these receptors initiates via these intracellular PRRS set in motion a series of events that has resulted in IRF3 and NF-κβ activation, both of which are required for the production of IFN-β and the release of chemokines that recruit immune cells to the site of infection [7, 9, 16].

## **4. How type I IFNs become central players in the connection between innate and acquired immune response**

 Type I IFNs are the dominant player of the connection between the innate and adaptive immune responses through the main interaction with antigen-presenting immune cells, such as dendritic cells (DCs), in particular, with plasmacytoid dendritic cells (pDCs) [6, 18, 21], which are precisely the major producers of type I IFNs producing up to a hundred to a thousand times more IFNs-α than other cell types [13, 14]. This is supported from *in vitro* experiments that have shown that type I IFN-treated DCs prime T cells *in vitro* more effectively [11, 12, 15].

## **5. How to calibrate host immune response to bacterial infections?**

 Calibrating host immune system for bacterial infections initiated as outlined above through the surface membrane conserved molecules organized in "patterns" such as peptidoglycan (PGN), lipopolysaccharide (LPS), and nucleic acid structures or pathogen-associated molecular patterns (PAMPs). Whereas, innate cells have the counterpart, "PRRS" (pattern recognition pathogen) [8, 10, 16], that automatically unlock the unspecificity of the type I IFNs production, the recognition of the "self" versus "nonself" [9], one PRRS for a particular type of PAMPs either bacterial, fungal, or virus; followed by a more general signalization route through Myd88 and IRFs (this could be also specific for each type of IFNs), and finally, NF-κβ translocation to the nucleus and thus IFNs production [8–10]. The synthesis of type I IFNs is not the job of a specialized cell type. However, an important distinction must be made between those cells that produce just enough type I IFNs to affect the local environment, and those produced by IFN-producing cells (IPCs) which could contribute to connect innate and adaptive immune responses more effectively. How much is produced or how much should be produced depends mostly on the tissue involved and the signal received, in particular, viral, bacterial [6, 18, 21]. Therefore, it is intriguing that all IFN-α proteins interact with the same receptor complex and have a spectrum of distinct effects, that goes from the specific antiviral capacity of individual IFN-α to differences in the activation of natural killer (NK) cells [16, 17]. Trying to understand why some types of IFNs, one temptative explanation could be, different temporal or spatial regulation of their expression, which might impact in the molecular calibration of the host immune response to viral or bacterial infections since TLR signaling targets (such as NF-κβ) and IFNAR signaling targets (such as STAT) converge at their promoters [10, 16]. Thus, it seems possible to think that it is the TLR4 signaling that arises as a key player for type I IFNs production by different cell types in response to Gram-negative pathogens. Several studies have in addition highlighted this point, some has been concentrated in the LPS effect [8, 16] on the type I IFNS induction, while others have focused in the gene that encode inducible oxide nitric synthase (iNOS), which is more evident once a bacterial signal through TLR, as demonstrated with Chlamydia spp. [8, 16]. Despite this gap in our

*Immunogenetic and Immunotherapy in Tuberculosis DOI: http://dx.doi.org/10.5772/intechopen.83030* 

 knowledge, the gene encoding iNOs is a paradigm for antimicrobial genes requiring type I IFN synthesis and expression downstream of TLR, implying a potential important role of type I IFN synthesis during nonviral infection. More recent infection studies that have investigated the mechanism behind this type I IFN effect demonstrated its importance in generating TNF-α, Il-1β, or bacterial signals (Chlamydia spp). IL-12-independent cellular immunity to *S. typhimurium*. This was attributed to the ability of type I IFNs to stimulate STAT-4 tyrosine phosphorylation in NK cells and Th1 cells. Together with IL-18 signals, this triggers expression of the IFN-alpha gene [8, 16]. In addition, it has been described that the induction of intrinsic immunity to kill bacteria or prevent their invasion and the regulation of chemokines, proinflammatory cytokines, and phagocytic cells. The mechanism by which IFN-α/β promotes host protective responses or susceptibility in bacterial pathogens is poorly defined and the factors that determine whether a response will be protective or pathogenic are not yet fully understood. However, it is well known that type I IFNs that are released during bacterial infection by IFNproducing cells (IPDCs) can cause the activation of signal transducer and activator of transcription 4 (STAT 4) in natural killer (NK) and T helper (TH1 cells) [5, 8, 10, 16]. In conjunction with interleukin 18 (IL-18)-derived signals, STAT-4 stimulates the expression of the IFN-alpha genes, which provide antibacterial immunity, such as macrophage activation [22, 23]. In addition, type I IFNs make important contributions to the maturation and activation of dendritic ells (DCs) [24], and in this way, influence antigen presentation, T cell activation, and the development of adaptive immune responses.

## **6. Dichotomy in the type I IFNs' action in bacterial infections**

 In contrast to viral infections, IFN-α/β can be protective or can have detrimental effects for the host during bacterial infections in a bacterium-specific manner, although less is known about the role of these. By one side, IFN-α/β-mediated signaling primes the production of interleukin-10 (IL-10), proinflammatory cytokines, and antimicrobial effector mechanism. But, IL-10 mediates a negative feedback loop, suppressing the production of proinflammatory cytokine, including IL-12, tumor necrosis factor (TNF), and IL-1 α/β cytokines that are key in the host resistance to bacterial infections. Moreover, some studies have addressed to decreased bacterial load and/or improved host survival in the absence of IFNα/β-mediated signaling. Thus, for example, IFN-α/β contributes to priming the host to clear the virus, while increasing host susceptibility to bacterial assault. Interestingly, under this scenario, IFN-α/β produced in response to infections is damaging to the host but would normally be protecting during a primary infection, i.e*.*, *S. pneumoniae* or *E. coli* [8, 10, 16]. This would imply that the circumstances of IFN-α/β production and action are crucial to determine host protection versus pathogenesis and highlight also the dichotomy role of IFN-α/β depending on the pathogen. These different issues have been pinpointed and clearly showed that, for example, on mycobacterial infections, there is a detrimental effect of type I IFNs in active TB patient, which showed in blood a remarkable transcriptional gene expression profile in neutrophils that correlated with extensive lesion in lung [25]. In a different cohort of patients from Africa, it was also found that this same result, the broad signature of IFN-α/β, could be found anywhere [25]. These findings have revealed the dark side of these cytokines that is—the ability to suppress host immune protective response by downregulating the Th1-type cellular immune responses (IFN-gamma IL12 production), iNOS3 synthesis while inducing IL-10. In summary, favorable or unfavorable effect can be determined by the infecting strain, the severity of infection, the stage of infection, and the interplay among the different immune effector mechanisms.

## **7. Signalization pathways of type I IFNs as an adjuvant**

 Adjuvants can stimulate innate immunity by interacting with specialized pattern recognition receptors (PRRs) including Toll-like receptors (TLRs) [26, 27] and nucleotide-binding oligomerization domain receptors [28]. These PRRs are immersed in the membrane surface of the antigen presenting such as DCs or macrophages, even in epithelial and B cells. Once this interaction is initiated, it is followed by serial reactions that lead to the production of proinflammatory cytokines and chemokines that will influence drastically the outcome of the host immune response. The shape of this response will be affected by initial stimulus, and therefore, the antigen-presenting cells (M1, M2) as well as the T cell population will adopt a state of differentiation (Th1/Th2/Th3) [29]. However, many cell types, including nonhematopoietic cells, express PRR and produce cytokines during innate immunity [30]. In conjunction, adjuvant action could be viewed as the contribution of cytokines milieu and the different cellular sources of them in order to initiate and potentiate immunity from the native polyclonal repertoire cells and molecules.

The role of IFN-I as natural immune adjuvants for commercial vaccines [18, 21, 31] was established by showing that either mucosal or intramuscular administration of influenza virus antigen-admixed IFN-I to mice enhances viral resistance and increased production of antiviral Ab [18, 21]. The adjuvant activity of IFN-I leads to potentiate the adaptive immune response by directly stimulating lymphocytes or activating DC that represents the critical antigen-presenting cells governing the fate of helper T cell responses [18, 21, 24, 25]. The immunity-promoting activity of IFN-I can result from a direct effect on T cells. In this situation, IFN-I acts as "third signal" of activation, helping to sustain survival of proliferating cells. IFN-I also supports Th1 differentiation, activation of STAT-4 signaling, and IFN-γ production [24, 25]. These activities are reminiscent of the biological effects of IL-12 and could have a role in the observed adjuvant type I IFN activities. Indeed, the variable need for IFN-I to act directly on T cells during activation and differentiation may thus arise from a similarly variable production of IL-12.

## **8. How type I IFNs shape the host immune system to antimycobacterial infection?**

Despite the wealth of studies, shaping the host immune response to bacterial infection is complex and still remains to be characterized. Type I IFNs can shape the antimycobacterial immunity by enhancing action of dendritic cells and monocytes, by promoting CD4+ and Cd8 T cell responses, by enhancing NK cell responses and B cell responses [8, 10, 16, 18, 21]. Type I IFNs (IFN-α/β) have a direct effect on the maturation of DCs, through increasing cell surface expression of MHC molecules as well as costimulatory molecules such as CD80 and CD86, leading to an augmented activation of T cells. Another effect of type I IFNs (IFN-α/β) is to promote the migration of DCs to lymph nodes through upregulating chemokine receptor expression thus promoting T cell activation. Moreover, direct downregulation of IFN-γR expression may not be the central mechanism by which IFN-αβ exerts their effects on IFN-γ activity [7–10, 18, 21–23], instead, in both mouse and human cells,

#### *Immunogenetic and Immunotherapy in Tuberculosis DOI: http://dx.doi.org/10.5772/intechopen.83030*

 it has been shown that IFN-αβ potently suppresses the ability of macrophages to upregulate antimycobacterial effector molecules and to restrict bacterial growth, in response to both *M. leprae* and *M tuberculosis.* The importance of this mechanism of action of IFN-αβ is further suggested by experiment using Ifngr 1-/- or Ifnar1-/ mice, which suggests that IFN-αβ contributes to host protection in the absence of the IFN-γ pathway [16, 17, 22, 23]. In another study, it was observed a natural mutation in the gene ISG15 in humans that conferred host-protective response mediated by type I IFNs (IFN-αβ) *to M. tuberculosis* infection [23]. No further studies were made. Similarly, it has been reported that IL-12p70 suppressed type I IFNs (IFN-αβ) during *M. tuberculosis* infection [27, 28, 32]. This suppression could result from the presence of IL-10, the downregulation of IFN-γR, and/or the induction of negative regulators of IFN-mediated signaling such as protein arginine methyltransferase-1(PRMT1) [9, 10, 21, 22]. Finally, IFN-αβ, possibly by influencing chemokine expression, has been shown to be involved in the generation and trafficking of *M. tuberculosis* permissive innate cells to the lungs in a mouse model thus contributing to the exacerbation of infection [8, 9, 26, 31, 32].

 Several human clinical studies have obtained favorable assessment of using aerosolized IFN-α as adjuvant therapy for patients with tuberculosis [33]. However, it has been shown that there is a TB reactivation during IFN-alpha treatment for hepatitis D infection [33]. In a different study, it has been also demonstrated that in active TB patients, there is a correlation between the extent of lung lesion with the transcriptional signature of type I IFNs in blood, in particular, in neutrophils [25]. This was also found in a cohort of Africa and Indonesia. These findings implied that the type I IFNs are common broad signature and strengthened the role of these cytokines in the pathogenesis of TB [8, 9, 25, 26, 34]. Indeed, seminal work by Giacomini et al., [24] have demonstrated that IFN-β improves *M. bovis* BCG vaccine immunogenic capacity by exerting a strong influence of DCs maturation, throughout enhancing costimulatory molecules such as CD86, CD83, and therefore, increased IL-12 which will act on macrophage killing activities [18, 20, 29, 30]. Later on, further studies by Mayer-Babier et al. [34] have demonstrated that the action of type I IFNs in tuberculosis could reside in the pathways of IL-1β, arachidonic acids, prostaglandins, and iNOs. Active TB patients showed an increased production of these molecules. This constitutes the first cue for a clinical therapeutic target of TB [34]. In more recent work by us, we found that type I IFNs action, in particular, interferon alpha, could exert its action in conjunction with *M. bovis* BCG vaccine that potentially could be signaling through Toll-like receptor and/or tentative through IFN-R1, leading to a protective antimycobacterial immune response, i.e., Th1-type cytokines and so far to IL-17 and IL23 production [35–37].

*Immunogenetics*

## **Author details**

Gloria Guillermina Guerrero Manriquez Immunobiology Laboratory, Science Biological Unit, Autonome University of Zacatecas, Zacatecas, Mexico

\*Address all correspondence to: gloguerrero9@gmail.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Immunogenetic and Immunotherapy in Tuberculosis DOI: http://dx.doi.org/10.5772/intechopen.83030* 

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## *Edited by Nima Rezaei*

Because genetic factors can impact immune responses, and immunogenetic associations serve as a predictor of disease development and as a biological indicator of disease progression, the study of immunogenetics is important to basic genetics and immunology, as well as to translational and individualized medicine.Tis book addresses a few but important issues on the subject of immunogenetics. First, it will review the role that human leukocyte antigen molecules play in the immune system and then take into consideration the efectiveness of Western bloting for the detection of immunologic proteins. Te book will discuss studies on the immunogenetics of cancer and tuberculosis followed by implications for immunotherapy. Working separately, the book will also provide evidence that the application of immunogenetics has improved our understanding of brain and behavior disorders.

Published in London, UK © 2019 IntechOpen © nnorozoff / iStock