Toll-Like Receptors, Keys of the Innate Immune System

*Alaa Fadhel Hassan*

#### **Abstract**

Toll-like receptors (TLRs) are members of the integral glycoproteins family, which are consist of intracellular and endoplasmic domains. TLRs are widely distributed in body tissues and expressed by immune and nonimmune cells. They are able to identify pathogens that cause cell injury and distinguish them from harmless microbes, and pathogenic nucleic acids as their binding ligand. Upon binding to their ligands, TLRs first underwent conformational changes; either forming homodimers or heterodimers, starting signaling pathways involve adaptor molecules utilization and then signal transduction through either myeloid differential (MyD)-88 dependent or independent pathways. Ending with activation of several transcription factors (TF) and release of pro-inflammatory cytokines (CK) and Type I interferons (IFN) and initiation of inflammation. TLRs are involved in almost all-inflammatory processes due to underlying disorders and diseases, which made them interesting targets for therapeutic development, via the synthesis of different agonists, antagonists, and even naturalized antibodies.

**Keywords:** Innate immune response, Toll-like receptors, Myeloid differential88, Cluster differential 14, Lymphocyte antigen 96 & Pro-inflammatory cytokines

#### **1. Introduction**

Our start point is that: inflammation is known pathogenesis of different pathophysiological conditions and diseases affecting different body tissues whether acute or chronic. Every inflammation involves an immune response -innate and adaptive- that started with specific receptors called recognition receptors to identify stimuli/damage signal, activation of consequence inflammatory pathway/cascade, the release of inflammatory markers, and recruitment of inflammatory immune cells [1].

The innate immune response is initiated by either endogenous ligands acting as damage signals known as the damage-associated molecular pattern (DAMPs), or exogenous pathogenic ligands-that are accurately portion of the pathogenic microorganism- lead to the same fate; damage signals throughout pathogen-associated molecular patterns (PAMPs) [2]. These patterns alter the body of the cell and cause tissue injuries leading to massive necrosis that release intracellular component into surrounding, these components activate TLRs [3, 4]. These processes, which are both the mechanism and the net results of inflammations, infections, or ischemic injuries cause more, harm than the initial causes itself by improper stimulation of the immune response [3, 4].

TLRs are a family of pattern recognition receptors (PPR), which also involves nucleotide oligomerization domain (NOD)-like receptors (NLR) and retinoic acidinducible gene I (RIG-I)-like receptors (RLR). They are located on cell membrane/ surface and nucleus, are responsible for the detection/recognition of the pathogen or intracellular damaged derived molecular signals to start immune response [1, 2].

These complicated inflammatory processes induced by the immune system are the "Classical typical scenario" involved in the majority of ischemic events, cancers, infectious and inflammatory diseases [4]. For further information about the immune system, Video 1 (https://youtu.be/8mEnyBdsrr8) can be shown on Armando Hasudungan YouTube channel [2] that would explain the innate immunity link with TLRs.

#### **2. Toll-like receptors**

TLRs are PRR family involves 13 members that exist in mammals with 10 members detected in the human genome [5, 6], depending on their similar morphology with Toll. Toll is a gene product that participate in both embryonic polarity development and adult fly -antimicrobial response of the species *Drosophila melanogaster* [6, 7]. A 1996 study of this gene product linked the loss/gain of function to the insect's susceptibility and immunologic response to fungal infections; increasing the temptation to seek for the amino acids sequence of the genome. This lead to the final identification of toll-like receptors in 1998 [4].

#### **2.1 Toll-like receptors tissue expression and cellular distribution**

TLRs are expressed in almost all body tissues involved in immunologic response as well as those exposed to external environments like the spleen, blood, lung & gastrointestinal tract [4, 8]. The particular cellular expression involves innate and adaptive immunity as well as different nonimmune cells. TLRs cellular expression involves the white blood cells "the sentinel of the innate immune response": microphages (MΦ) & mast cell (MC) "innate immune response keys", dendritic cells (DCs) (primarily pathogenic detector of the adaptive immune response) [4, 6, 8, 9], endothelial cells, epithelial cells, fibroblast, glial cells, astrocytes, oligodendrocytes, etc. [1, 5, 8, 10].

Cellular expression of TLRs family members largely variable and mainly depends on the presence of active infections [8]; according to the same source, as ex., bacterial product & pro-inflammatory cytokines can induce the expression of TL3 while IL-10 blocks TLR4 expression. It has been found that TLR2 expression is more specifically involved in the gram-positive bacteria signaling [8]. TLRs are located either primarily to immune cell plasma membrane phospholipids including TLR 1, 2, 4, 5, 6, & 11 [3, 4, 8]; Or located at the endosomal and lysosomal phospholipids where their extracellular domain (ECD) and its ligand-binding site project into the interior of the organelles like TLR 3, 7, 8, 9, 10 and 13 [2, 3, 10, 11].

#### **2.2 Toll-like receptors biochemistry and Structure**

TLRs are a type I integral transmembrane glycoprotein family of very conserved structure [5, 7], consist of 700–1100 amino acids [2, 4]. Their structure, shown in **Figure 1** consist of 2 domains: an ECD that recognize ligands, consist of repetitive motifs rich with leucine and an intracellular domain (ICD) –called cytoplasmicthat maintain inflammatory signal consequence, the last consist of interleukin (IL)-1 receptor region called Toll/IL receptor (TIR) domain [12, 13].

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structures.

*3.1.1 PAMPs*

8, and 9 [14].

**Figure 1.**

*Toll-Like Receptors, Keys of the Innate Immune System DOI: http://dx.doi.org/10.5772/intechopen.97502*

**3. Toll-like receptors family members**

*homology model is based on TLR2-TIR structure (PDB ID: 1fyw) [12].*

be shown at Armando Hasudungan YouTube channel [18].

**3.1 Toll-like receptors binding ligands**

TLRs involves 13 family members that exist in mammals with 10 members detected in the human genome [5, 6]. Human TLRs amino acids sequence allow a subfamily classification into the TLR2, TLR3, TLR4, TLR5, and TLR9 subfamilies. The TLR2 subfamily involves TLR1, 2, 6, and 10; the TLR9 subfamily involves TLR7,

*A representative structure of TLR. The conserved structural features of all TLRs consist of three critical components: (1) leucine-rich repeat (LRR) motif; (2) transmembrane helix; (3) intracellular TIR domain. The LRR structure is based on the model of TLR1-TLR2 heterodimer (Protein Data Bank, PDB, ID: 2z7x) interacting with six triacylated-lipopeptides, Pam3CysSerLys4 (Pam3CSK4), whereas the TIR domain* 

TLRs members can form homodimers/heterodimers among their same protein family or associates with an "outside TLR family" protein; both formations contribute to their structural and functional diversity [4]. Homodimers are formed by TLR4 while TLR members 1, 2, and 6 like TLR1/2 or TLR2/6 dimers form heterodimers [2, 3, 15–17]. TLRs members, their dimerization, cellular distribution, ligands, induced signaling pathway, and product are shown in **Table 1**; for further information about TLRs, Video 2 (https://youtu.be/8mEnyBdsrr8) about TLR overview can

TLRs family members can recognize two types of associated molecular patterns as their ligands, derived from pathogens or damaged organelles damaged

PAMPs derived from pathogen [5, 19]; like gram-negative bacterial lipopolysaccharides (LPS), gram-positive bacterial lipoteichoic acid (LTA) and peptidoglycan

*Toll-Like Receptors, Keys of the Innate Immune System DOI: http://dx.doi.org/10.5772/intechopen.97502*

**Figure 1.**

*Innate Immunity in Health and Disease*

immunity link with TLRs.

**2. Toll-like receptors**

final identification of toll-like receptors in 1998 [4].

astrocytes, oligodendrocytes, etc. [1, 5, 8, 10].

**2.2 Toll-like receptors biochemistry and Structure**

(IL)-1 receptor region called Toll/IL receptor (TIR) domain [12, 13].

**2.1 Toll-like receptors tissue expression and cellular distribution**

TLRs are expressed in almost all body tissues involved in immunologic response as well as those exposed to external environments like the spleen, blood, lung & gastrointestinal tract [4, 8]. The particular cellular expression involves innate and adaptive immunity as well as different nonimmune cells. TLRs cellular expression involves the white blood cells "the sentinel of the innate immune response": microphages (MΦ) & mast cell (MC) "innate immune response keys", dendritic cells (DCs) (primarily pathogenic detector of the adaptive immune response) [4, 6, 8, 9], endothelial cells, epithelial cells, fibroblast, glial cells,

Cellular expression of TLRs family members largely variable and mainly depends on the presence of active infections [8]; according to the same source, as ex., bacterial product & pro-inflammatory cytokines can induce the expression of TL3 while IL-10 blocks TLR4 expression. It has been found that TLR2 expression is more specifically involved in the gram-positive bacteria signaling [8]. TLRs are located either primarily to immune cell plasma membrane phospholipids including TLR 1, 2, 4, 5, 6, & 11 [3, 4, 8]; Or located at the endosomal and lysosomal phospholipids where their extracellular domain (ECD) and its ligand-binding site project into the interior of the organelles like TLR 3, 7, 8, 9, 10 and 13 [2, 3, 10, 11].

TLRs are a type I integral transmembrane glycoprotein family of very conserved structure [5, 7], consist of 700–1100 amino acids [2, 4]. Their structure, shown in **Figure 1** consist of 2 domains: an ECD that recognize ligands, consist of repetitive motifs rich with leucine and an intracellular domain (ICD) –called cytoplasmicthat maintain inflammatory signal consequence, the last consist of interleukin

TLRs are a family of pattern recognition receptors (PPR), which also involves nucleotide oligomerization domain (NOD)-like receptors (NLR) and retinoic acidinducible gene I (RIG-I)-like receptors (RLR). They are located on cell membrane/ surface and nucleus, are responsible for the detection/recognition of the pathogen or intracellular damaged derived molecular signals to start immune response [1, 2]. These complicated inflammatory processes induced by the immune system are the "Classical typical scenario" involved in the majority of ischemic events, cancers, infectious and inflammatory diseases [4]. For further information about the immune system, Video 1 (https://youtu.be/8mEnyBdsrr8) can be shown on Armando Hasudungan YouTube channel [2] that would explain the innate

TLRs are PRR family involves 13 members that exist in mammals with 10 members detected in the human genome [5, 6], depending on their similar morphology with Toll. Toll is a gene product that participate in both embryonic polarity development and adult fly -antimicrobial response of the species *Drosophila melanogaster* [6, 7]. A 1996 study of this gene product linked the loss/gain of function to the insect's susceptibility and immunologic response to fungal infections; increasing the temptation to seek for the amino acids sequence of the genome. This lead to the

**136**

*A representative structure of TLR. The conserved structural features of all TLRs consist of three critical components: (1) leucine-rich repeat (LRR) motif; (2) transmembrane helix; (3) intracellular TIR domain. The LRR structure is based on the model of TLR1-TLR2 heterodimer (Protein Data Bank, PDB, ID: 2z7x) interacting with six triacylated-lipopeptides, Pam3CysSerLys4 (Pam3CSK4), whereas the TIR domain homology model is based on TLR2-TIR structure (PDB ID: 1fyw) [12].*

#### **3. Toll-like receptors family members**

TLRs involves 13 family members that exist in mammals with 10 members detected in the human genome [5, 6]. Human TLRs amino acids sequence allow a subfamily classification into the TLR2, TLR3, TLR4, TLR5, and TLR9 subfamilies. The TLR2 subfamily involves TLR1, 2, 6, and 10; the TLR9 subfamily involves TLR7, 8, and 9 [14].

TLRs members can form homodimers/heterodimers among their same protein family or associates with an "outside TLR family" protein; both formations contribute to their structural and functional diversity [4]. Homodimers are formed by TLR4 while TLR members 1, 2, and 6 like TLR1/2 or TLR2/6 dimers form heterodimers [2, 3, 15–17]. TLRs members, their dimerization, cellular distribution, ligands, induced signaling pathway, and product are shown in **Table 1**; for further information about TLRs, Video 2 (https://youtu.be/8mEnyBdsrr8) about TLR overview can be shown at Armando Hasudungan YouTube channel [18].

#### **3.1 Toll-like receptors binding ligands**

TLRs family members can recognize two types of associated molecular patterns as their ligands, derived from pathogens or damaged organelles damaged structures.

#### *3.1.1 PAMPs*

PAMPs derived from pathogen [5, 19]; like gram-negative bacterial lipopolysaccharides (LPS), gram-positive bacterial lipoteichoic acid (LTA) and peptidoglycan


**139**

**Figure 2.**

*Toll-Like Receptors, Keys of the Innate Immune System DOI: http://dx.doi.org/10.5772/intechopen.97502*

**4. Toll-like receptors signaling pathway**

deoxyribonucleic acid (DNA) [20, 21].

*3.1.2 DAMPs*

tors NF-

**4.1 Co-receptors**

mation control [6, 24].

(PGN), mycobacterial lipopeptides, yeast zymosan, viral and bacterial ribonucleic acid (RNA), and unmethylated cytosine phosphate guanine containing- (CpG)

DAMPs damaged organelles structures, extracellular matrix, cytosolic and nuclear proteins, Heat shock protein-60 (HSP-60) and HSP-70, hyaluronic acid fragments, and free fatty acids (FFA) [5, 22, 23]. They cause activation of the innate and inflammatory immune responses, epithelial regeneration, and sterile inflam

Upon TLRs recognition and binding to their ligands, they undergo conforma

tional changes, dimerization as well as interaction with adaptor molecules passing series of intracellular signal transduction pathways that involve transcription fac

Co –receptors involved in TLRs signalling include Cluster differential 14 (CD14) and Lymphocyte antigen 96 (MD-2). Both have a major role in TLR4 activation after LPS recognition. CD14 is a glycophosphatidylinositol attached

*Signaling pathways of TLR. Surface and endosomal TLRs bind to adaptor molecules and co-receptors. Signal through Myd88 dependent/independent pathway ending with proinflammatory CK or type I IFN [12].*

nitric oxide (NO), CK- like tumour necrosis factor-alpha (TNF-

chemokines (CC), and type I IFN [15, 21, 25, 26]. As shown in **Figure 2**

κB, IRFs, and mitogen-activated protein kinase (MAPK) activation. These pathways finally resulting in the secretion of pro-inflammatory mediators including




β,

α), IL-6 & IL-1

.

*Toll-Like Receptors, Keys of the Innate Immune System DOI: http://dx.doi.org/10.5772/intechopen.97502*

(PGN), mycobacterial lipopeptides, yeast zymosan, viral and bacterial ribonucleic acid (RNA), and unmethylated cytosine phosphate guanine containing- (CpG) deoxyribonucleic acid (DNA) [20, 21].

#### *3.1.2 DAMPs*

*Innate Immunity in Health and Disease*

**138**

**TLRs** TLR1+

Cell surface

TLR2

TLR2+

Cell surface

TLR6

TLR3 TLR4

Cell surface/

endosomes

Mo, M

IE

TLR5 TLR7 TLR8

Endosomes

Mo, M

TLR9

Endosomes

Mo, M

TLR10

Endosomes

Mo, M

*Mo: monocytes, M*

**Table 1.**

*TLRs cellular expression, binding ligands, signal adaptor & production [2].*

Φ, DC

Φ, DC, B, T

Φ, DC, MC

Endosomes

ssRNA

ssRNA

Imidazoquinolin-es (R848)

Guanosine analogues (Loxoribine)

ssRNA,

ssRNA Chromatin IgG complex

MyD88 MyD88

IC

IC, type1 IFN

MyD88

IC, type1 IFN

Imidazoquinolines (R848)

CpG DNA

CpG ODNs

profilin-like proteins

*Φ: macrophages, DC: dendritic cells, MC: Mast cells, B: B cells, T: T cells, IE: Intestinal epithelium, IC: Inflammatory cytokines [2]*

Mo, M

Φ, DC. B

Cell surface

Flagellin

Mo, M

Φ, DC, IE

Φ, DC, MC,

Endosomes

dsRNA (poly (I:C))

tRNA, siRNA

Lipopolysaccharides (LPS)

Paclitaxel

B, T, NK, DC

Mo, M

Φ, MC, B

Mo, M

Φ, DC, B

**Immune Cell Expression**

**PAMPs** Tri-acylated lipoproteins (Pam3CSK4)

Peptidoglycans, Lipopolysaccharides

Diacylated lipoproteins

(FSL-1)

**DAMPs** (TLR2 DAMPs listed below)

Heat Shock Proteins

(HSP 60, 70, Gp96)

High mobility group proteins (HMGB1)

Proteoglycans

(Versican, Hyaluronic Acid fragments)

mRNA

TRIF TRAM, TRIF

IC, type1 IFN

TIRAP, MyD88

Mal

IC, type1 IFN

tRNA

Heat Shock Proteins

(HSP22, 60, 70,72, Gp96)

High mobility group proteins (HMGB1)

Proteoglycans

(Versican, Heparin sulfate,

Hyaluronic Acid fragments)

Fibronectin, Tenascin-C

MyD88 MyD88

IC, type1 IFN

IC

**Signal Adaptor**

TIRAP, MyD88,

IC

Mal

TIRAP, MyD88,

IC

Mal

**Production**

DAMPs damaged organelles structures, extracellular matrix, cytosolic and nuclear proteins, Heat shock protein-60 (HSP-60) and HSP-70, hyaluronic acid fragments, and free fatty acids (FFA) [5, 22, 23]. They cause activation of the innate and inflammatory immune responses, epithelial regeneration, and sterile inflammation control [6, 24].

#### **4. Toll-like receptors signaling pathway**

Upon TLRs recognition and binding to their ligands, they undergo conformational changes, dimerization as well as interaction with adaptor molecules passing series of intracellular signal transduction pathways that involve transcription factors NF-κB, IRFs, and mitogen-activated protein kinase (MAPK) activation. These pathways finally resulting in the secretion of pro-inflammatory mediators including nitric oxide (NO), CK- like tumour necrosis factor-alpha (TNF-α), IL-6 & IL-1β, chemokines (CC), and type I IFN [15, 21, 25, 26]. As shown in **Figure 2**.

#### **4.1 Co-receptors**

Co –receptors involved in TLRs signalling include Cluster differential 14 (CD14) and Lymphocyte antigen 96 (MD-2). Both have a major role in TLR4 activation after LPS recognition. CD14 is a glycophosphatidylinositol attached

#### **Figure 2.**

*Signaling pathways of TLR. Surface and endosomal TLRs bind to adaptor molecules and co-receptors. Signal through Myd88 dependent/independent pathway ending with proinflammatory CK or type I IFN [12].*

protein expressed on innate immune cells as macrophage and monocytes that function as co-receptor for both cell surface & endosomal expressed TLRs. Lymphocyte antigen 96 (MD-2), which is a cell membrane glycoprotein associated specifically with TLR4 ECD, and expressed at myeloid and endothelial cells [6, 13, 21, 26, 27].

#### **4.2 Adaptor proteins and kinase molecules**

TLRs signaling pathways involves four main adaptor protein molecules: MyD88, TIR domain-containing adaptor protein/MyD88 adaptor-like molecules (TIRAP) also called MAL, TIR domain-containing adaptor protein inducing interferon-β (TRIF), and TRIF related adaptor molecule (TRAM) [13, 21, 28]. TLRs signaling pathways involves activation of five TIR containing adaptor kinase molecules, like IL-1 receptor-associated kinase (IRAK) -1 and 4, TNF receptor-associated factor-6 (TRAF6), serine/threonine binding kinase (TBK)-1, MAPK, and inhibitor of kappa-B (IκB) kinase (IKK) [13, 28].

#### **4.3 Transcription factors**

There are three transcription factors involved in the TLRs signalling pathway including NF-κB, AP1, and IRF. NF-κB is an intracellular pleiotropic protein complex; it is responsible for gene regulation of proinflammatory CK, CC, adhesion molecules, and cell cycle/survival regulating proteins as cyclin D1 and B cell lymphoma 2 (Bcl-2). AP1 is a dimer of both protein Jun and Fos families; that is associated with cell replication and survival regulation. Finally, the IRFs protein regulating IFNs, are responsible for signal stimulation via MyD88independent/ TRIF pathway [6, 13].

#### **4.4 Intracellular signaling pathways**

There are two intracellular signalling pathways for TLRs involve MyD88 dependent/& MyD88-independent also called (TRIF-dependant) signal transduction pathway.

#### *4.4.1 MyD88-dependent pathway*

It is utilized by all TLRs but not TLR3 [21, 29]. This pathway activates the IRAKs, TRAF6, transforming growth factor (TGF)-β-activated kinase (TAK)-1 and the IKK complex [15]. It causes the nuclear translocation of NF-κB and adaptor protein-1 (AP1) [28, 30], and ends with the secretion of CK like IL-6, IL-10, IL-12 & TNF-α [16, 29]. MyD88 also stimulate the classical extracellular signal-regulated kinases (MAPK/ERK), phosphoinositide-3 (PI3), and Jun (N)terminal kinase (JNK) which stimulate the AP1 signalling pathway, and induce the interferon regulatory factor-7 (IRF7) ending with the release of type-I IFN or co-stimulatory molecules associated with the antimicrobial response by endosomal TLRs 3, 7, 8 and 9 [13, 29, 31, 32].

#### *4.4.2 MyD88-independent pathway*

The main pathway of TLR3 and 4, involve TRIF signalling pathway activation which involves TRAF6 activation, results in inositol triphosphate-3 (IP3) phosphorylation and induction of IFN-β gene expression as well as activation of TRAF6 [21, 29].

**141**

of IL-1β [35, 38].

*Toll-Like Receptors, Keys of the Innate Immune System DOI: http://dx.doi.org/10.5772/intechopen.97502*

dependent pathway [15, 33].

and independent pathways [11, 21, 29].

signalling for these receptors" [5, 13].

**4.5 Unique pathways**

description section.

mation itself [35, 36].

Surprisingly the same outcome was obtained from plasmatoid dendritic cells (pDCs) stimulated by TLR 7& 9 throughout the activation of the MyD88/IRF7

TLR4 further utilizes TIRAP to activate MyD88 and TRAM to bridge the TRIF activation, which means that TLR4 uniquely utilizes both the MYD88 dependent

As stated by S. Kiziltas et al. "TLR stimulation product is dependent on the nature of PAMPs, the activated TLR, the activated cell and the level of CK. Moreover, the chronically activated signalling pathway would possibly induce transcription of oncogenic factor; adding further complexation to the intracellular

TLRs play an important role in pathophysiological disorders due to their wide tissue distribution, their function as pattern recognition receptors that respond to variable bacterial and damage associated molecules, and involvement in multiple inflammatory signal pathways/& process all render TLRs being a major player in any inflammation-related disorder [4–6, 19, 22, 23, 34]. In addition, analysis of TLRs gene polymorphism in human disorders revealed an increased risk of bacterial infection and sepsis as an example [34]. This section is a shortcut or summary to TLR involvement in different pathophysiological disorders rather than a full

**5.1 Toll-like receptors and pathophysiology of inflammatory oxidative stress**

Inflammation is a common etiology of many disorders and disease including ischemic injuries, microbial infections, diabetes, arthritis and cancer [3, 4, 35]; still, any inflammatory process is triggered by damage signal recognized by pattern receptors and induce activation of signaling pathways leading to the production of pro-inflammatory markers and activation of immune cells [35]. These processes also induce the release of free radicals (FR) such as reactive oxygen species (ROS) and the activation of hypoxia-inducible transcription factor-1 (HIF1), causing tissue stress and reduced tissue oxygen status, so-called tissue hypoxia. Hypoxia is believed to be a hallmark as well as a key trigger of inflam-

Under normal conditions HIF1-α subunit (the inducible form of the heterodimer protein HIF-1 transcription complex) [35], is controlled by hydroxylation of proline residue via prolyl hydroxylase enzyme, and breaking down via proteasome. However under inflammatory conditions LPS activate TLRs that stimulates nicotine amide adenine dinucleotide phosphate (NADPH) oxidase (Nox)-associated crosstalk with the MAPK signaling pathways [36, 37], that causes proinflammatory CK & markers production thus increasing mitochondrial FR release like ROS causing more and more tissue stress. That causes HIF1- α activation; here HIF1- α protein inactivation process will be inhibited due to proline consumption, leading to HIF1-α accumulation in MΦ, DCs and other non-immune cells that exposed to hypoxia/ & non-hypoxic damage signals [38]. Furthermore, this would induce metabolic reprogramming of mitochondrial respiration causing succinate release, and production

**5. Toll-like receptors and pathophysiological disorders**

Surprisingly the same outcome was obtained from plasmatoid dendritic cells (pDCs) stimulated by TLR 7& 9 throughout the activation of the MyD88/IRF7 dependent pathway [15, 33].

#### **4.5 Unique pathways**

*Innate Immunity in Health and Disease*

**4.2 Adaptor proteins and kinase molecules**

kappa-B (IκB) kinase (IKK) [13, 28].

**4.4 Intracellular signaling pathways**

*4.4.1 MyD88-dependent pathway*

**4.3 Transcription factors**

TRIF pathway [6, 13].

and 9 [13, 29, 31, 32].

TRAF6 [21, 29].

*4.4.2 MyD88-independent pathway*

tion pathway.

cells [6, 13, 21, 26, 27].

protein expressed on innate immune cells as macrophage and monocytes that function as co-receptor for both cell surface & endosomal expressed TLRs. Lymphocyte antigen 96 (MD-2), which is a cell membrane glycoprotein associated specifically with TLR4 ECD, and expressed at myeloid and endothelial

TLRs signaling pathways involves four main adaptor protein molecules: MyD88, TIR domain-containing adaptor protein/MyD88 adaptor-like molecules (TIRAP) also called MAL, TIR domain-containing adaptor protein inducing interferon-β (TRIF), and TRIF related adaptor molecule (TRAM) [13, 21, 28]. TLRs signaling pathways involves activation of five TIR containing adaptor kinase molecules, like IL-1 receptor-associated kinase (IRAK) -1 and 4, TNF receptor-associated factor-6 (TRAF6), serine/threonine binding kinase (TBK)-1, MAPK, and inhibitor of

There are three transcription factors involved in the TLRs signalling pathway

including NF-κB, AP1, and IRF. NF-κB is an intracellular pleiotropic protein complex; it is responsible for gene regulation of proinflammatory CK, CC, adhesion molecules, and cell cycle/survival regulating proteins as cyclin D1 and B cell lymphoma 2 (Bcl-2). AP1 is a dimer of both protein Jun and Fos families; that is associated with cell replication and survival regulation. Finally, the IRFs protein regulating IFNs, are responsible for signal stimulation via MyD88independent/

There are two intracellular signalling pathways for TLRs involve MyD88 dependent/& MyD88-independent also called (TRIF-dependant) signal transduc-

It is utilized by all TLRs but not TLR3 [21, 29]. This pathway activates the IRAKs, TRAF6, transforming growth factor (TGF)-β-activated kinase (TAK)-1 and the IKK complex [15]. It causes the nuclear translocation of NF-κB and adaptor protein-1 (AP1) [28, 30], and ends with the secretion of CK like IL-6, IL-10, IL-12 & TNF-α [16, 29]. MyD88 also stimulate the classical extracellular signal-regulated kinases (MAPK/ERK), phosphoinositide-3 (PI3), and Jun (N)terminal kinase (JNK) which stimulate the AP1 signalling pathway, and induce the interferon regulatory factor-7 (IRF7) ending with the release of type-I IFN or co-stimulatory molecules associated with the antimicrobial response by endosomal TLRs 3, 7, 8

The main pathway of TLR3 and 4, involve TRIF signalling pathway activation which involves TRAF6 activation, results in inositol triphosphate-3 (IP3) phosphorylation and induction of IFN-β gene expression as well as activation of

**140**

TLR4 further utilizes TIRAP to activate MyD88 and TRAM to bridge the TRIF activation, which means that TLR4 uniquely utilizes both the MYD88 dependent and independent pathways [11, 21, 29].

As stated by S. Kiziltas et al. "TLR stimulation product is dependent on the nature of PAMPs, the activated TLR, the activated cell and the level of CK. Moreover, the chronically activated signalling pathway would possibly induce transcription of oncogenic factor; adding further complexation to the intracellular signalling for these receptors" [5, 13].

#### **5. Toll-like receptors and pathophysiological disorders**

TLRs play an important role in pathophysiological disorders due to their wide tissue distribution, their function as pattern recognition receptors that respond to variable bacterial and damage associated molecules, and involvement in multiple inflammatory signal pathways/& process all render TLRs being a major player in any inflammation-related disorder [4–6, 19, 22, 23, 34]. In addition, analysis of TLRs gene polymorphism in human disorders revealed an increased risk of bacterial infection and sepsis as an example [34]. This section is a shortcut or summary to TLR involvement in different pathophysiological disorders rather than a full description section.

#### **5.1 Toll-like receptors and pathophysiology of inflammatory oxidative stress**

Inflammation is a common etiology of many disorders and disease including ischemic injuries, microbial infections, diabetes, arthritis and cancer [3, 4, 35]; still, any inflammatory process is triggered by damage signal recognized by pattern receptors and induce activation of signaling pathways leading to the production of pro-inflammatory markers and activation of immune cells [35]. These processes also induce the release of free radicals (FR) such as reactive oxygen species (ROS) and the activation of hypoxia-inducible transcription factor-1 (HIF1), causing tissue stress and reduced tissue oxygen status, so-called tissue hypoxia. Hypoxia is believed to be a hallmark as well as a key trigger of inflammation itself [35, 36].

Under normal conditions HIF1-α subunit (the inducible form of the heterodimer protein HIF-1 transcription complex) [35], is controlled by hydroxylation of proline residue via prolyl hydroxylase enzyme, and breaking down via proteasome. However under inflammatory conditions LPS activate TLRs that stimulates nicotine amide adenine dinucleotide phosphate (NADPH) oxidase (Nox)-associated crosstalk with the MAPK signaling pathways [36, 37], that causes proinflammatory CK & markers production thus increasing mitochondrial FR release like ROS causing more and more tissue stress. That causes HIF1- α activation; here HIF1- α protein inactivation process will be inhibited due to proline consumption, leading to HIF1-α accumulation in MΦ, DCs and other non-immune cells that exposed to hypoxia/ & non-hypoxic damage signals [38]. Furthermore, this would induce metabolic reprogramming of mitochondrial respiration causing succinate release, and production of IL-1β [35, 38].

In dendritic cells, TLRs cause further stabilization of HIF1-α via release of NF-κB, which would further increase glucose uptake and render shifting of mitochondrial respiration to the anaerobic glycolytic pathway due to the increased oxygen demand versus the decreased supply [35, 36]. Finally result in disruption of the normal function of DCs, the primary pathogenic detector of the adaptive immune response; which undergo cellular maturation upon TLRS activation that results in further expression of co-stimulatory molecules, further production of pro-inflammatory CK & CC, and migration to lymph node so to present antigens to naïve T-cells [4, 35]. All these scenarios would further amplify the existing inflammation and tissue damage [35].

HIF1-α is a transcription factor that responsible for cellular adaptive responses after exposure to injury/stress environment, including maintenance like controlling angiogenesis to improve blood vessel formation, shifting cellular mitochondria respiration to anaerobic glycolysis through improving cellular survival and cellular adhesion in oxidative stress environment's [36]. In addition, it is the major controller of phagocytes bactericidal capacity, and involved in myeloid cell-mediated inflammation, and is an essential factor for inhibition of myeloid cell apoptosis induced by LPS. The last point made it an important factor also in the TLR4 signaling pathway [36, 38]. HIF1-α function as a double-edged sword, that mediate cellular adaptive to stress but progress disease status by the same time [38].

#### **5.2 Toll-like receptors important role in the pathophysiological disorder**

#### *5.2.1 Central nervous system*

TLRs are expressed in various central nervous system (CNS) cells predominantly in neurons, astrocytes, resident microglia, cerebral microvasculature, plexuses choroid, and leptomeninges. They are associated with the detection of- and regulated by central DAMPs [33]. TLR4 is further upregulated centrally by glutamate via N-methyl-D-aspartate (NMDA) dependent mechanism and peripherally by noradrenaline/β2 receptor, & corticotrophin-releasing factor. TLRs play an important role in restoring central homeostasis, physiology of stress-sensitive behaviour after injuries or diseases as multiple sclerosis, Alzheimer's, and stroke [33].

In the experimental model of CNS, stress exposure revealed mRNA upregulation and activation of TLRs in the brain frontal cortex after the stress is involved in the loss of neuronal plasticity and survival depending on the activation of NF-κB induced ROS production. Also resultant bacterial translocation from the gut to the systemic circulation and other organs such as the liver, spleen, and mesenteric lymph nodes; These circulating gram-negative bacteria are the major source of LPS, which can activate brain TLR4 through multiple pathways, including a neuroinflammatory response. This is partially explained by the theory known as leaky gut [11, 33].

In another experimental model of neurogenesis, TLR3 & 4 were found to act as down regulators, TLR3 deletion/loss of function was also linked to improved cognitive function. The same reference state an opposed case in viral meningitis when TLR3 & 9 recruitment help to decrease neuronal injury and localize infection area and in Alzheimer disease where TLR2, 4, 5, 7 & 9 were suggested to improve disease progression by inhibiting amyloid plaque accumulation [1].

#### *5.2.2 Respiratory system*

TLR is thought to play a considerable role in several respiratory disorders starting from allergic rhinitis ending with severe inflammatory disorders like acute respiratory distress syndrome (ARDS), through their activation by the causative

**143**

*Toll-Like Receptors, Keys of the Innate Immune System DOI: http://dx.doi.org/10.5772/intechopen.97502*

involved in-the causative inflammation [37].

same ligand of TLR4 itself. [2, 3, 37].

failure, atherosclerosis & myocarditis [9, 27, 39–41].

*5.2.3 Cardiovascular system*

*5.2.4 Gastrointestinal system*

inflammations derived by pulmonary oedema, trauma, sepsis & even drug overdose [9, 37]. In allergic rhinitis TLR2, 3, & 4 were found to be both upregulated by- and

TLR2 has the mainstay of involvement & determination in respiratory allergic disease due to considerable genetic variation. In asthma, an experimental study shows TLR2 induction by synthetic Pam3Cys triggers immune response & disease severity [37]. While in acute lung injury (ALI) & ARDS, TLR2 was found to be activated by Toll interacting protein (Tollip) [14]. TLR4 was found to increase asthmatics severity & prevalence in paediatrics. TLR4 genetic polymorphism affects cluster differentials (CD)41–251 regulatory T cells (Tregs) which are activated by LPS, the

An experimental model of doxorubicin and hydrogen peroxide-induced cardiac injury showed TLR2 to be involved in cardio myocytes apoptosis, besides TLR2 targeting suggested to be protective in septic cardiomyopathy [1]. In addition, murine models revealed cardiac tissue expression of TLR4 increased after hypertension, myocardial ischemia, maladaptive left ventricular hypertrophy, and angiotensin II (AngII) infusion participating in vascular remodelling & stiffness, endothelial dysfunction, increase myocardial infarction (MI) size & susceptibility. While Human studies revealed the same in patients with unstable angina, MI, heart

TLR4 expression & signalling was increased in patients' monocytes during attacks of unstable angina & MI [37]. In the experimental model & human vascular inflammation, TLR4 was found to increase the production of CK, CC as well as increase TLR2 expression. In the early stage of the atherosclerotic lesion, TLR4 mRNA protein was detected & MyD88 -the mainstay of TLR signalling pathwaygene deficiency was linked to decrement in CK, CC & lipid content production, as well as in atherosclerotic lesion size. The same reference stated that TLR2 genetic polymorphism was linked to increased coronary artery stenosis, while TLR7 & 8

was involved in cardiac inflammation caused by the Coxsackie virus [37].

holic liver injuries, ischemia/reperfusion injury, and carcinoma [13, 28].

acid, causing steatosis, necrosis, and hepatic congestion [16].

The liver is the major organ that deals with gut-derived endotoxin, exposed by portal circulation [13, 42]. This continuous exposure would trigger frequent activation of the hepatic innate immune system; which contributes to the induction of inflammation in acute hepatic injuries, which means involvement of TLRs in the induction of inflammation [13]. Pathogenic suppression/& inhibition of TLRs found to mediates chronic hepatic injuries/disorders like hepatitis, fibrosis, alco-

In Paracetamol human hepatotoxicity, endogenous chemical injury derives extracellular matrix (ECM) the ligand that activates TLR4 to release TNF-α, induce inducible nitric oxide synthase (iNOS), peroxynitrite, glutathione depletion, so that will amplify immune response, sequestering leukocytes, increase serum hyaluronic

Hepatitis viral nucleic acid & proteins are the ligands detected by TLR3, 7, 8, & 9. Starting with hepatitis B virus (HBV), in vitro activation of TLR1, 2, 3, 4, 5, 6, 7, 8, & 9 result in the release of IFN which inhibit HBV DNA replication and RNA transcription. Whilst HBV itself downregulates the expression of TLR1, 2, 4, & 6, this limits their antiviral effect or even renders them nugatory [28]. This downregulation of TLRs is attributed to the presence of HBV e antigen (HBeAg) *Toll-Like Receptors, Keys of the Innate Immune System DOI: http://dx.doi.org/10.5772/intechopen.97502*

inflammations derived by pulmonary oedema, trauma, sepsis & even drug overdose [9, 37]. In allergic rhinitis TLR2, 3, & 4 were found to be both upregulated by- and involved in-the causative inflammation [37].

TLR2 has the mainstay of involvement & determination in respiratory allergic disease due to considerable genetic variation. In asthma, an experimental study shows TLR2 induction by synthetic Pam3Cys triggers immune response & disease severity [37]. While in acute lung injury (ALI) & ARDS, TLR2 was found to be activated by Toll interacting protein (Tollip) [14]. TLR4 was found to increase asthmatics severity & prevalence in paediatrics. TLR4 genetic polymorphism affects cluster differentials (CD)41–251 regulatory T cells (Tregs) which are activated by LPS, the same ligand of TLR4 itself. [2, 3, 37].

#### *5.2.3 Cardiovascular system*

*Innate Immunity in Health and Disease*

mation and tissue damage [35].

*5.2.1 Central nervous system*

In dendritic cells, TLRs cause further stabilization of HIF1-α via release of NF-κB, which would further increase glucose uptake and render shifting of mitochondrial respiration to the anaerobic glycolytic pathway due to the increased oxygen demand versus the decreased supply [35, 36]. Finally result in disruption of the normal function of DCs, the primary pathogenic detector of the adaptive immune response; which undergo cellular maturation upon TLRS activation that results in further expression of co-stimulatory molecules, further production of pro-inflammatory CK & CC, and migration to lymph node so to present antigens to naïve T-cells [4, 35]. All these scenarios would further amplify the existing inflam-

HIF1-α is a transcription factor that responsible for cellular adaptive responses after exposure to injury/stress environment, including maintenance like controlling angiogenesis to improve blood vessel formation, shifting cellular mitochondria respiration to anaerobic glycolysis through improving cellular survival and cellular adhesion in oxidative stress environment's [36]. In addition, it is the major controller of phagocytes bactericidal capacity, and involved in myeloid cell-mediated inflammation, and is an essential factor for inhibition of myeloid cell apoptosis induced by LPS. The last point made it an important factor also in the TLR4 signaling pathway [36, 38]. HIF1-α function as a double-edged sword, that mediate cellular adaptive to stress but progress disease status by the same time [38].

**5.2 Toll-like receptors important role in the pathophysiological disorder**

in neurons, astrocytes, resident microglia, cerebral microvasculature, plexuses choroid, and leptomeninges. They are associated with the detection of- and regulated by central DAMPs [33]. TLR4 is further upregulated centrally by glutamate via N-methyl-D-aspartate (NMDA) dependent mechanism and peripherally by noradrenaline/β2 receptor, & corticotrophin-releasing factor. TLRs play an important role in restoring central homeostasis, physiology of stress-sensitive behaviour after

In the experimental model of CNS, stress exposure revealed mRNA upregulation and activation of TLRs in the brain frontal cortex after the stress is involved in the loss of neuronal plasticity and survival depending on the activation of NF-κB induced ROS production. Also resultant bacterial translocation from the gut to the systemic circulation and other organs such as the liver, spleen, and mesenteric lymph nodes; These circulating gram-negative bacteria are the major source of LPS, which can activate brain TLR4 through multiple pathways, including a neuroinflammatory response. This is partially explained by the theory known as leaky gut [11, 33].

In another experimental model of neurogenesis, TLR3 & 4 were found to act as down regulators, TLR3 deletion/loss of function was also linked to improved cognitive function. The same reference state an opposed case in viral meningitis when TLR3 & 9 recruitment help to decrease neuronal injury and localize infection area and in Alzheimer disease where TLR2, 4, 5, 7 & 9 were suggested to improve disease

TLR is thought to play a considerable role in several respiratory disorders starting from allergic rhinitis ending with severe inflammatory disorders like acute respiratory distress syndrome (ARDS), through their activation by the causative

injuries or diseases as multiple sclerosis, Alzheimer's, and stroke [33].

progression by inhibiting amyloid plaque accumulation [1].

TLRs are expressed in various central nervous system (CNS) cells predominantly

**142**

*5.2.2 Respiratory system*

An experimental model of doxorubicin and hydrogen peroxide-induced cardiac injury showed TLR2 to be involved in cardio myocytes apoptosis, besides TLR2 targeting suggested to be protective in septic cardiomyopathy [1]. In addition, murine models revealed cardiac tissue expression of TLR4 increased after hypertension, myocardial ischemia, maladaptive left ventricular hypertrophy, and angiotensin II (AngII) infusion participating in vascular remodelling & stiffness, endothelial dysfunction, increase myocardial infarction (MI) size & susceptibility. While Human studies revealed the same in patients with unstable angina, MI, heart failure, atherosclerosis & myocarditis [9, 27, 39–41].

TLR4 expression & signalling was increased in patients' monocytes during attacks of unstable angina & MI [37]. In the experimental model & human vascular inflammation, TLR4 was found to increase the production of CK, CC as well as increase TLR2 expression. In the early stage of the atherosclerotic lesion, TLR4 mRNA protein was detected & MyD88 -the mainstay of TLR signalling pathwaygene deficiency was linked to decrement in CK, CC & lipid content production, as well as in atherosclerotic lesion size. The same reference stated that TLR2 genetic polymorphism was linked to increased coronary artery stenosis, while TLR7 & 8 was involved in cardiac inflammation caused by the Coxsackie virus [37].

#### *5.2.4 Gastrointestinal system*

The liver is the major organ that deals with gut-derived endotoxin, exposed by portal circulation [13, 42]. This continuous exposure would trigger frequent activation of the hepatic innate immune system; which contributes to the induction of inflammation in acute hepatic injuries, which means involvement of TLRs in the induction of inflammation [13]. Pathogenic suppression/& inhibition of TLRs found to mediates chronic hepatic injuries/disorders like hepatitis, fibrosis, alcoholic liver injuries, ischemia/reperfusion injury, and carcinoma [13, 28].

In Paracetamol human hepatotoxicity, endogenous chemical injury derives extracellular matrix (ECM) the ligand that activates TLR4 to release TNF-α, induce inducible nitric oxide synthase (iNOS), peroxynitrite, glutathione depletion, so that will amplify immune response, sequestering leukocytes, increase serum hyaluronic acid, causing steatosis, necrosis, and hepatic congestion [16].

Hepatitis viral nucleic acid & proteins are the ligands detected by TLR3, 7, 8, & 9. Starting with hepatitis B virus (HBV), in vitro activation of TLR1, 2, 3, 4, 5, 6, 7, 8, & 9 result in the release of IFN which inhibit HBV DNA replication and RNA transcription. Whilst HBV itself downregulates the expression of TLR1, 2, 4, & 6, this limits their antiviral effect or even renders them nugatory [28]. This downregulation of TLRs is attributed to the presence of HBV e antigen (HBeAg) during acute infection. About hepatitis C (HCV), its core protein activates TLR 1, 2, 4 & 6, which are supposed to produce antiviral IFNs as well as increased hepatic inflammation. The same effect is presumed by TLR 3 & 4 in HBV is achieved here to produce IFN-β [28].

In alcoholic liver disease (ALD), alcohol mainstay effects are to increase gut mucosal permeability to LPS, modification of gut flora, reducing endotoxin clearance rate, and increasing hepatic endotoxin level [16]. These scenarios lead to higher expression of TLR1, 2, 4, 6 & 9 by both parenchymal and non-parenchymal cells, activating their pathway and release of inflammatory mediators, this process observed in the chronic alcohol experimental model [28, 29]. While a patient with cirrhosis expresses a high level of TNF-α, IL-1β, & IL-6, as well as chronic endotoxemia, recurrent bacterial infection [16]. Finally, the process of hepatic regeneration depends on the interplay between the immune system and non-parenchymal cell, which involves activation of TLRs/MyD88 pathway, here the bulky activation of TLRs, would inversely affect the regeneration process, which indicates that the extent of such activation is essential for hepatic regeneration. TLR2, 4 & 9 reported no important role in liver regeneration process [28, 43].

Both human patients and experimental models of diabetes linked the active TLR to the progression of diabetes complication throughout the activation of NF-κB signalling in adipose tissue MΦ due to high level of plasma FFA associated with obesity & diabetes type 2 (T2DM) [44].

In vivo & in-vitro studies performed by Zhang N. et al. revealed that TLR 2 & 4 activation in insulin target tissues as the liver, adipose tissue & immune cells linked them with insulin resistance. The first suggests that high TLRs loss of function or genetic modification protects against high FFA level resulted from large mass adipose tissue secreting non-esterified free fatty acids & reduction of their clearance/ oxidation which disturbs gut permeability to LPs [45].

TLR4 resultant inflammation associated with activation IKK, MAPK, JNK, and p38 pathways would further increase insulin receptor substrate-1 (IRS1) serine phosphorylation thus decrease insulin receptor's signal transduction [31, 45]. Furthermore, TLR4-MyD88 signalling pathway activation was suggested throughout developmental researches for several anti-hyperlipidemic medications, while TLR1, 2, 3 & 7 were triggering both host immune defence and/autoimmune response that aggravate diabetic state [37].

#### *5.2.5 Urinary system*

TLRs expression in renal tube epithelial lining render their activation to be essential in renal vascular remodelling, endothelial dysfunction in multiple renal disorders like acute kidney injury (AKI), solid organ transplant, glomerulonephritis, ischemic/reperfusion injury (I/R injury) & diabetic renal disorders [27, 44]. Experimental streptozocin induced diabetic model revealed podcytopathy & fibrosis regression after TLR4 knocking out, as they are expressed by podocytes & decreased diabetic nephropathy after TLR2 knocking out [46, 47]. TLR4 gene polymorphism was linked to prostate cancer among gene clusters of TLR1, 6 & 10 [37].

#### **6. Toll-like receptors as therapeutic targets**

TLRs, as the primary receptor for many ligands that trigger innate & adaptive immune response, with complex signaling pathways involving many adaptor

**145**

*Toll-Like Receptors, Keys of the Innate Immune System DOI: http://dx.doi.org/10.5772/intechopen.97502*

chloroquine [28, 34].

**6.1 Toll-like receptors 1 and 2**

alleviate disease progression [9].

**6.2 Toll-like receptor 3**

**6.3 Toll like receptor 4**

molecules & co-receptors seem interesting for therapeutic target development. Synthetic agonist, antagonist and even naturalized antibodies could modify TLRs signaling to make them attractive targets for the management of different inflammatory disease. For example at 2013, Savva and Roger enlisted around 32 clinical trials at different phases for TLRs agonist/antagonist agent for the management of sepsis and infectious disease, these trials include even the antimalarial old agent

TLR1/2 heterodimers were found to be increased in patients with atherosclerotic lesions, while administration of TLR1/2 agonist aggravates disease status, also TLR2 inhibition was suggested as diabetes and cardiovascular disorders therapy besides statins & thiazolidinedione by anti-inflammatory action [9]. Pam2/3CSK4 TLR2 ligands covalently linked to CD8+ or B-cell epitopes associated peptides were found to enhance therapeutic response in tumour models, by stimulating TLR2 induced T-cell activation [15]. A 3 component carbohydrate-based cancer vaccine involved TLR2 activator that mediates humoral immune response against tumour-induced glycopeptide antigens by affecting the maturation of cellular component of the innate immune system (DC & natural killer cells), furthermore cancer treatment with chimers of anti-tumour antibodies and small molecule agonist of TLR2 would

Since high synovial expression of TLR3 in RA patients was found, one scenario

Various TLR4 antagonist was developed as a therapeutic agent, starting with the peptide P13- an inhibitor of TIR domain signalling pathway- that was found to ameliorate inflammatory response and improve surviving in a TLR4-mediated hepatic injury of murine model [16]. In addition, Lipid A mimetics E5564 and CRX526 bind to TLR4-MD2 complex showing valuable inhibition of pro-inflammatory cytokine IL-1 and TNF-α production in LPS treated animal models as well as septic shock patients in phase III clinical trial [9, 16, 29]. TLR4 inhibition was suggested as the scenario for treatment of thrombosis, atherosclerosis & vascular restenosis throughout coating TLR4 or MyD88 with inhibitory compound, small molecule antagonist, then by giving viral vectors that express antisense gene to TLR4 RNA [9], and finally TLR4/MD2/anti-Human IgG (Fc specific) (IgG-Fc) fusion protein inhibitor of NF-κB and JNK activation provides interesting biologic therapy for liver fibrosis, alcoholic and non-alcoholic steatohepatitis by decreasing IL-6 and

for rheumatoid arthritis and possibly bone malignancy is to inhabit the TLR3 pathway via the RNA synthetic analogue Polyinosine-polycytidylic acid (poly (I:C)

that affect monocyte –osteoclast cellular differentiation [9].

monocyte Chemoattractant Protein-1(MCP-1) production [16].

HBV vaccine in treating viral hepatitis [13, 15, 16].

Another TLR4-synergizer Fc/fusion protein and TL4 ligand α-1 acid glycoprotein were found to inhibit LPS-induced activation of hepatic MΦ by blocking the triggering receptor expressed on myeloid cells-1 (TREM1), and boosting the anti-inflammatory immune response. Other theoretically interesting scenarios involving the I.V administration of monophosphoryl lipid A derivatives as 2 adult molecules & co-receptors seem interesting for therapeutic target development. Synthetic agonist, antagonist and even naturalized antibodies could modify TLRs signaling to make them attractive targets for the management of different inflammatory disease. For example at 2013, Savva and Roger enlisted around 32 clinical trials at different phases for TLRs agonist/antagonist agent for the management of sepsis and infectious disease, these trials include even the antimalarial old agent chloroquine [28, 34].

#### **6.1 Toll-like receptors 1 and 2**

*Innate Immunity in Health and Disease*

to produce IFN-β [28].

during acute infection. About hepatitis C (HCV), its core protein activates TLR 1, 2, 4 & 6, which are supposed to produce antiviral IFNs as well as increased hepatic inflammation. The same effect is presumed by TLR 3 & 4 in HBV is achieved here

In alcoholic liver disease (ALD), alcohol mainstay effects are to increase gut mucosal permeability to LPS, modification of gut flora, reducing endotoxin clearance rate, and increasing hepatic endotoxin level [16]. These scenarios lead to higher expression of TLR1, 2, 4, 6 & 9 by both parenchymal and non-parenchymal cells, activating their pathway and release of inflammatory mediators, this process observed in the chronic alcohol experimental model [28, 29]. While a patient with cirrhosis expresses a high level of TNF-α, IL-1β, & IL-6, as well as chronic endotoxemia, recurrent bacterial infection [16]. Finally, the process of hepatic regeneration depends on the interplay between the immune system and non-parenchymal cell, which involves activation of TLRs/MyD88 pathway, here the bulky activation of TLRs, would inversely affect the regeneration process, which indicates that the extent of such activation is essential for hepatic regeneration. TLR2, 4 & 9 reported

Both human patients and experimental models of diabetes linked the active TLR

In vivo & in-vitro studies performed by Zhang N. et al. revealed that TLR 2 & 4 activation in insulin target tissues as the liver, adipose tissue & immune cells linked them with insulin resistance. The first suggests that high TLRs loss of function or genetic modification protects against high FFA level resulted from large mass adipose tissue secreting non-esterified free fatty acids & reduction of their clearance/

TLR4 resultant inflammation associated with activation IKK, MAPK, JNK, and p38 pathways would further increase insulin receptor substrate-1 (IRS1) serine phosphorylation thus decrease insulin receptor's signal transduction [31, 45].

Furthermore, TLR4-MyD88 signalling pathway activation was suggested throughout developmental researches for several anti-hyperlipidemic medications, while TLR1, 2, 3 & 7 were triggering both host immune defence and/autoimmune response that

TLRs expression in renal tube epithelial lining render their activation to be essential in renal vascular remodelling, endothelial dysfunction in multiple renal disorders like acute kidney injury (AKI), solid organ transplant, glomerulonephritis, ischemic/reperfusion injury (I/R injury) & diabetic renal disorders [27, 44]. Experimental streptozocin induced diabetic model revealed podcytopathy & fibrosis regression after TLR4 knocking out, as they are expressed by podocytes & decreased diabetic nephropathy after TLR2 knocking out [46, 47]. TLR4 gene polymorphism was linked to prostate cancer among gene clusters of

TLRs, as the primary receptor for many ligands that trigger innate & adaptive immune response, with complex signaling pathways involving many adaptor

to the progression of diabetes complication throughout the activation of NF-κB signalling in adipose tissue MΦ due to high level of plasma FFA associated with

no important role in liver regeneration process [28, 43].

oxidation which disturbs gut permeability to LPs [45].

**6. Toll-like receptors as therapeutic targets**

obesity & diabetes type 2 (T2DM) [44].

aggravate diabetic state [37].

*5.2.5 Urinary system*

TLR1, 6 & 10 [37].

**144**

TLR1/2 heterodimers were found to be increased in patients with atherosclerotic lesions, while administration of TLR1/2 agonist aggravates disease status, also TLR2 inhibition was suggested as diabetes and cardiovascular disorders therapy besides statins & thiazolidinedione by anti-inflammatory action [9]. Pam2/3CSK4 TLR2 ligands covalently linked to CD8+ or B-cell epitopes associated peptides were found to enhance therapeutic response in tumour models, by stimulating TLR2 induced T-cell activation [15]. A 3 component carbohydrate-based cancer vaccine involved TLR2 activator that mediates humoral immune response against tumour-induced glycopeptide antigens by affecting the maturation of cellular component of the innate immune system (DC & natural killer cells), furthermore cancer treatment with chimers of anti-tumour antibodies and small molecule agonist of TLR2 would alleviate disease progression [9].

#### **6.2 Toll-like receptor 3**

Since high synovial expression of TLR3 in RA patients was found, one scenario for rheumatoid arthritis and possibly bone malignancy is to inhabit the TLR3 pathway via the RNA synthetic analogue Polyinosine-polycytidylic acid (poly (I:C) that affect monocyte –osteoclast cellular differentiation [9].

#### **6.3 Toll like receptor 4**

Various TLR4 antagonist was developed as a therapeutic agent, starting with the peptide P13- an inhibitor of TIR domain signalling pathway- that was found to ameliorate inflammatory response and improve surviving in a TLR4-mediated hepatic injury of murine model [16]. In addition, Lipid A mimetics E5564 and CRX526 bind to TLR4-MD2 complex showing valuable inhibition of pro-inflammatory cytokine IL-1 and TNF-α production in LPS treated animal models as well as septic shock patients in phase III clinical trial [9, 16, 29]. TLR4 inhibition was suggested as the scenario for treatment of thrombosis, atherosclerosis & vascular restenosis throughout coating TLR4 or MyD88 with inhibitory compound, small molecule antagonist, then by giving viral vectors that express antisense gene to TLR4 RNA [9], and finally TLR4/MD2/anti-Human IgG (Fc specific) (IgG-Fc) fusion protein inhibitor of NF-κB and JNK activation provides interesting biologic therapy for liver fibrosis, alcoholic and non-alcoholic steatohepatitis by decreasing IL-6 and monocyte Chemoattractant Protein-1(MCP-1) production [16].

Another TLR4-synergizer Fc/fusion protein and TL4 ligand α-1 acid glycoprotein were found to inhibit LPS-induced activation of hepatic MΦ by blocking the triggering receptor expressed on myeloid cells-1 (TREM1), and boosting the anti-inflammatory immune response. Other theoretically interesting scenarios involving the I.V administration of monophosphoryl lipid A derivatives as 2 adult HBV vaccine in treating viral hepatitis [13, 15, 16].

#### **6.4 Toll-like receptors 5 and 7**

One possible scenario for cancer immunotherapy involved TLR5 binding to flagellin that can turn the tolerogenic DCs into active antigen-presenting cells (APC) [9].

Isatoribine, a TLR7 agonist administered I.V was found to decrease viral load with a moderate adverse effect profile in HCV patients. In addition, IGS-9620 that was experimentally assessed on the HBV animal model was found to decrease HBVs antigen (HBsAg) level in serum, HBV viral load as well as IFN-α in dose dependent-manner [15, 29]. Note that some TLR7 targeting therapies were approved by Food and Drug Administration (FDA) like imiquimod, TLR7-immune response modifier that was approved since 1997 for treatment of superficial skin malignant melanoma & genital warts by increasing cellular production of CK like IFN, IL-6 & TNF [9].

#### **6.5 Toll-like receptor 9**

Selective TLR9 agonists like 1018 ISS (immunomodulatory sequences) that contain repeated CpG motifs were found to modulate the TLR9 signalling pathway involved in HBV infection and have been tested in phase III clinical trials. Another agonist IMO-2055 was under assessment in 2011 for oncologic disease as well as IMO-2125 which was found to maintain the high level of IFN was under assessment as a possible therapy to HCV patients. The TLR9 intracellular signalling inhibitors ST2825 and RO0884 designed to block IRAK1 &4/MyD88 singling pathway caused inhibition of the NF-κB, IL-1β, and TNF-α activation as well as decreased hepatic IL-6 secretion [9, 15, 29].

#### **7. Conclusions**

Medical and pharmacological development is focusing on the molecular level, in all aspects including analytical, physiological, pharmacological and even genetic aspects. Understanding immune response is thus important subject, furthermore, the target receptors which damage signals bind to, their signaling pathways end products will tell what possible immune response happened to human body. Tolllike receptors are those targets, the family of integral transmembrane glycoprotein expressed intracellularly or at cellular surface, considered main component and link between innate and adaptive immune response, which can induce signaling pathways involving four main adaptor molecules that initiate divaricated steps ending with inflammatory cytokines. These pathways could be involved in any inflammatory process/disorders and thus seems interesting targets for pharmacological intervention; all these steps bring us back to the bullet that explodes all these events in the body, the immune system.

#### **Conflict of interest**

The author declares no conflict of interest.

#### **Notes/thanks/other declarations**

After precious Thanks to Almighty and Merciful GOD, I would like to express my thanks and gratitude to Professor G. H. Majeed who made it possible to me to

**147**

*Toll-Like Receptors, Keys of the Innate Immune System DOI: http://dx.doi.org/10.5772/intechopen.97502*

**Abbreviations and nomenclature**

AKI Acute kidney injury ALD Alcoholic liver disease ALI Acute lung injury AngII Angiotensin II AP-1 Adaptor protein-1 APC Antigen-presenting cell

Bcl-2 B cell lymphoma 2 CC Chemokine

DC Dendritic cell

CD14 Cluster differential 14 CK Pro-inflammatory cytokines CpG Cytosine phosphate guanine

ECD Extracellular domain ECM Extracellular matrix FFA Free fatty acids FR Free radicals HBeAg HBV e antigen HBsAg HBV s antigen

HIF1 Hypoxia-inducible factor-1

I/R injury Ischemic/reperfusion injury

IgG-Fc Anti-Human IgG (Fc specific) IKK Inhibitor of kappa-B (IκB) kinase

iNOS Inducible nitric oxide synthase

IRAK IL-1 receptor-associated kinase IRF Interferon regulatory factor IκB Inhibitor of kappa-B JNK Jun (N)terminal kinase LPS Lipopolysaccharides LRR Leucine-rich repeat LTA Lipoteichoic acid

MAPK Mitogen-activated protein kinase MAPK/ERK Extracellular signal-regulated kinases

MCP-1 Monocyte Chemoattractant Protein-1

IP3 Inositol triphosphate-3

MD-2 Lymphocyte antigen 96 MI Myocardial infarction mRNA Messenger ribonucleic acid MyD88 Myeloid differential88

HSP Heat shock protein

ICD Cytoplasmic domain IFN Type-I interferon

IL Interleukin

MC Mast cell

ARDS Acute respiratory distress syndrome

DAMPs Damage-associated molecular pattern

accomplish this situation and reach you, my readers. Also to the magnificent group who introduced me to the world of toll-like receptors: professor Dr. Abduladheem Y. Abbood Al-Barrak, professor Dr. Bassim I. Mohammad, Dr. Samer Fadhel Hassan,

Dr. Asma A. Swadi, and Dr. Huda J. Merza, and sure my own family.

*Toll-Like Receptors, Keys of the Innate Immune System DOI: http://dx.doi.org/10.5772/intechopen.97502*

*Innate Immunity in Health and Disease*

**6.4 Toll-like receptors 5 and 7**

IFN, IL-6 & TNF [9].

**6.5 Toll-like receptor 9**

IL-6 secretion [9, 15, 29].

in the body, the immune system.

**Notes/thanks/other declarations**

The author declares no conflict of interest.

**Conflict of interest**

**7. Conclusions**

One possible scenario for cancer immunotherapy involved TLR5 binding to flagellin that can turn the tolerogenic DCs into active antigen-presenting cells (APC) [9]. Isatoribine, a TLR7 agonist administered I.V was found to decrease viral load with a moderate adverse effect profile in HCV patients. In addition, IGS-9620 that was experimentally assessed on the HBV animal model was found to decrease HBVs antigen (HBsAg) level in serum, HBV viral load as well as IFN-α in dose dependent-manner [15, 29]. Note that some TLR7 targeting therapies were

approved by Food and Drug Administration (FDA) like imiquimod, TLR7-immune response modifier that was approved since 1997 for treatment of superficial skin malignant melanoma & genital warts by increasing cellular production of CK like

Selective TLR9 agonists like 1018 ISS (immunomodulatory sequences) that contain repeated CpG motifs were found to modulate the TLR9 signalling pathway involved in HBV infection and have been tested in phase III clinical trials. Another agonist IMO-2055 was under assessment in 2011 for oncologic disease as well as IMO-2125 which was found to maintain the high level of IFN was under assessment as a possible therapy to HCV patients. The TLR9 intracellular signalling inhibitors ST2825 and RO0884 designed to block IRAK1 &4/MyD88 singling pathway caused inhibition of the NF-κB, IL-1β, and TNF-α activation as well as decreased hepatic

Medical and pharmacological development is focusing on the molecular level, in all aspects including analytical, physiological, pharmacological and even genetic aspects. Understanding immune response is thus important subject, furthermore, the target receptors which damage signals bind to, their signaling pathways end products will tell what possible immune response happened to human body. Tolllike receptors are those targets, the family of integral transmembrane glycoprotein expressed intracellularly or at cellular surface, considered main component and link between innate and adaptive immune response, which can induce signaling pathways involving four main adaptor molecules that initiate divaricated steps ending with inflammatory cytokines. These pathways could be involved in any inflammatory process/disorders and thus seems interesting targets for pharmacological intervention; all these steps bring us back to the bullet that explodes all these events

After precious Thanks to Almighty and Merciful GOD, I would like to express my thanks and gratitude to Professor G. H. Majeed who made it possible to me to

**146**

accomplish this situation and reach you, my readers. Also to the magnificent group who introduced me to the world of toll-like receptors: professor Dr. Abduladheem Y. Abbood Al-Barrak, professor Dr. Bassim I. Mohammad, Dr. Samer Fadhel Hassan, Dr. Asma A. Swadi, and Dr. Huda J. Merza, and sure my own family.

## **Abbreviations and nomenclature**



#### **Video materials**

#### **Vedio 1. Immunology-Toll Like Receptors Overview**

YouTube video: [3] Armando Hasudungan, Immunology-Toll Like Receptors Overview [Internet. YouTube]. 2014. Available from: https://youtu. be/8mEnyBdsrr8

#### **Vedio 2. Toll Like Receptors Overview**

YouTube video: [18] Armando Hasudungan, Immunology - Toll Like Receptors Overview [Internet YouTube]. 2014. Available from: https://youtu.be/8mEnyBdsrr8

**149**

**Author details**

Alaa Fadhel Hassan

Department of Pharmacy, Al-Mahmoudiya General Hospital, Baghdad, Iraq

© 2021 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,

\*Address all correspondence to: alaa.alwazni@yahoo.co.uk

provided the original work is properly cited.

*Toll-Like Receptors, Keys of the Innate Immune System DOI: http://dx.doi.org/10.5772/intechopen.97502*

*Toll-Like Receptors, Keys of the Innate Immune System DOI: http://dx.doi.org/10.5772/intechopen.97502*

*Innate Immunity in Health and Disease*

NADPH nicotine amide adenine dinucleotide phosphate

NOD nucleotide oligomerization domain

PAMPs Pathogen-associated molecular patterns

NLR nucleotide oligomerization domain (NOD)-like receptors

RLR retinoic acid-inducible gene I (RIG-I)-like receptors

TAK Transforming growth factor (TGF)-β-activated kinase

TIRAP/MAL TIR domain-containing adaptor protein/MyD88 adaptor like

TRIF TIR domain-containing adaptor protein inducing interferon-β

YouTube video: [3] Armando Hasudungan, Immunology-Toll Like Receptors Overview [Internet. YouTube]. 2014. Available from: https://youtu.

YouTube video: [18] Armando Hasudungan, Immunology - Toll Like Receptors Overview [Internet YouTube]. 2014. Available from: https://youtu.be/8mEnyBdsrr8

MΦ Macrophage

NO Nitric oxide

Nox NADPH oxidase Pam3CSK4 Pam3CysSerLys4

PGN Peptidoglycan PI3 Phosphoinositide-3

T2DM Diabetes type 2

TIR Toll/IL-receptor

TLRs Toll-like receptors

Tregs Regulatory T cells

**Video materials**

be/8mEnyBdsrr8

pDCs Plasmatoid dendritic cells

ROS Reactive oxygen species

TF Transcription factors TGF Transforming growth factor

poly(I:C) Polyinosine-polycytidylic acid PRRs Pattern recognition receptors RIG-I retinoic acid-inducible gene I

TBK-1 Serine/threonine binding kinase

TNF-α Tumour necrosis factor-alpha Tollip Toll interacting protein

**Vedio 2. Toll Like Receptors Overview**

TRAF6 TNF receptor-associated factor-6 TRAM TRIF related adaptor molecule

TREM1 Triggering receptor expressed on myeloid cells-1

**Vedio 1. Immunology-Toll Like Receptors Overview**

**148**

#### **Author details**

Alaa Fadhel Hassan Department of Pharmacy, Al-Mahmoudiya General Hospital, Baghdad, Iraq

\*Address all correspondence to: alaa.alwazni@yahoo.co.uk

© 2021 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.

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Allavena, and Alberto Mantovani: Differential Expression and Regulation of Toll-Like Receptors (TLR) in Human Leukocytes: Selective Expression of TLR3 in Dendritic Cells1. The Hournal of Immunology Hune 1,2000, 164(11)5998-6004. DOI: 10.4049/ jimmunol.164.11.5998

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[12] Gao W, Xiong Y, Li Q and Yang H: Inhibition of Toll-Like Receptor Signaling as a Promising Therapy for Inflammatory Diseases: A Journey from Molecular to Nano Therapeutics.Front. Physiol. 8:508.2017. DOI: 10.3389/ fphys.2017.00508

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theoncologist.2015-0164.

single dose administration of Methotrexate for the treatment of ectopic pregnancy: A systematic review and meta-analysis. Reproductive BioMedicine Online. 2017Apr;34(4):383- 391. DOI: 10.1016/j.rbmo.2017.01.004

[18] Armando Hasudungan, Immunology - Toll Like Receptors Overview [Internet YouTube]. 2014. Available from: https://youtu.

[19] Zhang E, and Lu M: Toll-like receptor (TLR)-mediated innate immune responses in the control of hepatitis B virus (HBV) infection. Medical Microbiology and Immunity 2015;204:11-20. DOI: 10.1007/

[20] Min HS, Kim JE, Lee MH, Song HK,

Hyun YY, Han JY, Cha DR and Kang YS: Effects of toll-like receptor antagonist 4,5-dihydro-3-phenyl-5-isoxasole acetic acid on the progression of kidney disease in mice on high-fat diet. Kidney

Lee MJ, Lee JE, Kim HW, Cha JJ,

be/8mEnyBdsrr8

s00430-014-0370-1

immunol.21.120601.141126

*Toll-Like Receptors, Keys of the Innate Immune System DOI: http://dx.doi.org/10.5772/intechopen.97502*

[14] Kiyoshi Takeda, Tsuneyasu Kaisho, Shizuo Akira. Toll-Like Receptors. Annual Review of Immunology 2003 21:1, 335-376. DOI:10.1146/annurev. immunol.21.120601.141126

[15] Quetglas EG, Armuzzi A, Wigge S, Fiorino G, Barnscheid L, Froelich M, and Danese S. Review article: The pharmacokinetics and pharmaco dynamics of drugs used in inflammatory bowel disease treatment. European Journal of Clinical Pharmacology, 2015;71(7):773-99. DOI: doi. org/10.1007/s00228-015-1862-7

[16] Howard SC, McCormick J, Pui CH, Buddington RK, Harvey RD. Preventing and Managing Toxicities of High-Dose Methotrexate. Oncologist. 2016 Dec;21(12):1471-1482. Doi: 10.1634/ theoncologist.2015-0164.

[17] Yang C, Cai J, Geng Y. and Gao Y. Multiple-dose and double-dose versus single dose administration of Methotrexate for the treatment of ectopic pregnancy: A systematic review and meta-analysis. Reproductive BioMedicine Online. 2017Apr;34(4):383- 391. DOI: 10.1016/j.rbmo.2017.01.004

[18] Armando Hasudungan, Immunology - Toll Like Receptors Overview [Internet YouTube]. 2014. Available from: https://youtu. be/8mEnyBdsrr8

[19] Zhang E, and Lu M: Toll-like receptor (TLR)-mediated innate immune responses in the control of hepatitis B virus (HBV) infection. Medical Microbiology and Immunity 2015;204:11-20. DOI: 10.1007/ s00430-014-0370-1

[20] Min HS, Kim JE, Lee MH, Song HK, Lee MJ, Lee JE, Kim HW, Cha JJ, Hyun YY, Han JY, Cha DR and Kang YS: Effects of toll-like receptor antagonist 4,5-dihydro-3-phenyl-5-isoxasole acetic acid on the progression of kidney disease in mice on high-fat diet. Kidney

Research and Clinical Practice 33(2014)33-44. DOI: 10.1016/j. krcp.2013.11.022

[21] Takashima K, Matsunaga N, Yokshimatsu M, Hazeki K, Kaisho T, Uekata M, Hazeki O, Akira S, Lizawa Y, and Li M: Analysis of binding site for the novel small-molecule TLR 4 signal transduction inhibitor TAK242 and its therapeutic effect on mouse sepsis model. British Journal of Pharmacology. (2009),157,1250-1262. DOI: 10.1111/j.1476-5381.2009.00297.x

[22] Zhang Y, Peng W, Ao X, Dai H, Yuan L, Hung X, et al: TAK-242, a toll-like receptor 4 antagonist, protect against aldosterone-induced cardiac and renal injury. Plos ONE 2015, 10(11):e0142456. DOI: 10.1371/journal. pone.0142456

[23] Broering R, Lu M, and Schlaak F: Role of toll-like receptors in liver health and disease. Clinical Science Nov 2011,121(10),415-426. DOI: 10.1042/ CS20110065

[24] Hadi N, and Jabber H: Potential activity of GIT27 against renal ischemia reperfusion injury: An experimental study in male rats. Pathophysiology of Cell Injury Journal 2016;5(2):87-99. DOI: 10.18081/2378-5225-016-12187-99

[25] Carrascosa J.M., de la Cueva P., Ara M., Puig L., Bordas X., Carretero G. et al. Metotrexato en psoriasis moderadagrave: Revisiόn de la literature y recomendaciones de expert. Actas Dermosifiliogr. 2016.Apr,107(3): 194-206. DOI: 10.1016/j.ad.2015-10.005

[26] Granucci Francesca, Zanoni Ivan. Role of CD14 in host protection against infections and in metabolism regulation. Frontiers in Cellular and Infection Microbiology 3, 2013 pages 32. DOI=10.3389/fcimb.2013.00032

[27] Li M, Matsunaga N, Hazeki K, Nakamura K, Takashima K, Seya T,

**150**

*Innate Immunity in Health and Disease*

[1] Amene Saghazadeh and Nima Rezaei Introductory Chapter: Toll-Like Receptors, Toll-like Receptors. Nima Rezaei, editor. IntechOpen January

Allavena, and Alberto Mantovani: Differential Expression and Regulation of Toll-Like Receptors (TLR) in Human Leukocytes: Selective Expression of TLR3 in Dendritic Cells1. The Hournal

of Immunology Hune 1,2000, 164(11)5998-6004. DOI: 10.4049/

[9] Jezierska A, Kolosova IA, and Verin AD. Toll Like Receptors Signaling Pathways as a Target for Therapeutic Interventions. Curr Signal Transduct Ther. 2011,6(3):428-440. DOI: 10.2174/157436211797483930.

[10] Kawasaki T, Kawai T. Toll-like receptor signaling pathways. Front Immunol. 2014;5:461. Published 2014 Sep 25. DOI:10.3389/fimmu.2014.

[11] Chalasani N, Fontana RJ,

gastro.2008.09.011

fphys.2017.00508

of GIT 27 and TAK 242 on

liver-injury-149321031

Bonkovsky HL, Watkins PB, Davern T, Serrano J, Yang H, Rochon J; Drug Induced Liver Injury Network (DILIN). Causes, clinical features, and outcomes from a prospective study of druginduced liver injury in the United States. Gastroenterology. 2008 Dec;135(6): 1924-34, 1934.e1-4. DOI: 10.1053/j.

[12] Gao W, Xiong Y, Li Q and Yang H: Inhibition of Toll-Like Receptor Signaling as a Promising Therapy for Inflammatory Diseases: A Journey from Molecular to Nano Therapeutics.Front. Physiol. 8:508.2017. DOI: 10.3389/

[13] Hassan AF: Eavluation of the effects

AlaaAlwazni/evaluation-of-the-effectof-git-27-tak-242-on-druginduced-

methotrexate-induced liver injury [thesis] Al-Mustansiriyah University, College of Medicine; 2018. Available from: https://pt.slideshare.net/

jimmunol.164.11.5998

00461

15th 2020). DOI: 10.5772/

youtu.be/8mEnyBdsrr8

[Accessed: 2021-03-09]

[2] Invivogen Reviews Toll-Like Receptors [Internet]. 2012. Available from: http://www.invivogen.com/ review-tlr [Accessed: 2021-03-09]

[3] Armando Hasudungan, Immunology-Toll Like Receptors Overview [Internet. YouTube]. 2014. Available from: https://

[4] Christmas P. Toll-Like Receptors: Sensors that Detect Infection, Nature Education [Internet]. 2010. Available from: https://www.nature.com/scitable/ topicpage/toll-like-receptors-sensorsthat-detect-infection-14396559/

[5] Kiziltas S: Toll-like receptors in pathophysiology of liver disease. World Journal of Hepatology 2016;8(32):1354- 1369. DOI: 10.4254/wjh.v8.i32.1354

[6] Guo J, and Fridman S: Toll-like receptor 4 signaling in liver injury and hepatic fibrogenesis. Fibrogenesis & Tissue Repair 2010,3:21. DOI: 10.1186/1755-1536-3-21

[7] Matsunaga N, Tsuchimori N, Matsumoto T, and Li M: TAK-242 (Resatorvid), a small-molecule inhibitor of toll-like receptor (TLR) 4 signaling, binds selectively to TLR4 and interferes with interaction between TLR4 and its

adaptor molecules. Molecular

DOI: 10.1124/mol.110.068064

Pharmacology Journal 1,2011,79(1)34-4.

[8] Marta Muzio, Daniela Bosisio, Nadia Polentarutti, Giovanna D'amico, Antonella Stoppacciaro, Roberta Mancinelli, Cornelis van't Veer, Giselle Penton-Rol, Luigi P. Ruco, Paola

intechopen.88493

**References**

Hazeki O, Kitazaki T, and Lizawa Y. A novel cyclohexene derivative, ethyl(6R)- 6-[N(2-choro-4-fluorophenyl) sulfamoyl]cyclohex-1-ene-1-carboxylate (TAK-242), selectively inhibits toll-like receptor 4-mediated cytokine production through suppression of intracellular signaling. Molecular Pharmacology April1,2006,69(4)1288- 1295. DOI:10.1124/mol.105.019695

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[30] Coskun M, Steenholdt C, de Boer NK and Nielsen OH. Pharmacology and optimization of thiopurines and methotrexate in inflammatory bowel disease. Clinical pharmacokinetics (2016)55,257-74. DOI: 10.1007/ s40262-015-0316-9

[31] Kaplowitz N. (2004) Drug induced liver injury. Clinical Infectious disease 2004Mar:38(Suppl2):S44-8. DOI:10.1086/38446

[32] Bianchi G, Caporali R, Todoerti M, and Mattana P. Methotrexate and rheumatoid arthritis: Current evidence regarding subcutaneous versus oral routes of administration. ADVANCES IN THERAPY 2016;33:369-378. DOI: 10.1007/s12325-016-0295-8

[33] Roth AD and Lee MY. Idiosyncratic drug-induced liver injury (IDILI): Potential mechanisms and protective

assays. BioMed Research International 2017,2017:9176937,23pages. DOI: 10.1155/2017/9176937

[34] Savva A, and Roger T. Targeting toll-like receptors: promising therapeutic strategies for the management of sepsis-associated pathology and infectious diseases. Front Immunol. 2013 Nov 18;4:387. DOI:10.3389/fimmu.2013.00387

[35] Spirig R, Djafarzadeh S, Regueira T, Shaw SG, von Garnier C, et al. Effects of TLR Agonists on the Hypoxia-Regulated Transcription Factor HIF-1α and Dendritic Cell Maturation under Normoxic Conditions. PLOS ONE 2010 5(6): e10983. DOI: 10.1371/journal. pone.0010983

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[44] Fagone P, Mangano K, Mammana S, Pesce A, Pesce A, Caltabino R and Nicoletti F, et al. Identification of novel targets for the diagnosis and treatment of liver fibrosis. International Journal of Molecular Medicine 36:747-752,2015. DOI: 10.3892/ijmm.2015.2264

[45] Zhang N, Liang H, Farese RV, Li J, Musi N, Hussey SE. Pharmacological TLR 4 inhibition protects against acute and chronic fat-induced insulin resistance in rats. Plos ONE

10(7):e013575. DOI: 10.1371/journal.

Pathophysiologic basis, and the current and emerging therapies. EMJ Hepatol. 2014;1:99-107. Available from: https:// emj.emg-health.com/wp-content/ uploads/sites/2/2018/02/Acute-Liver-Failure-Pathophysiologic-Basis-And-

[46] Privitera G, Agarwal B, and Jalan R. Acute Liver Failure:

The-Current-And-Emerging-

acid on methotrexate-induced hepatotoxicity in rat. Journal of xenobiotics 2016;6:6092. DOI: doi. org/10.408/xeno.2016,6092

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[42] Singh D, Cho WC, and Upadhyay G. Drug-induced liver toxicity and prevention by herbal antioxidants: An overview. Frontiers In Physiology 1.6:363. DOI: 10.3389/

fphys.2015-00363

15. DOI: 10.1097/01. JAA.0000515554.91731.82. *Toll-Like Receptors, Keys of the Innate Immune System DOI: http://dx.doi.org/10.5772/intechopen.97502*

clinicians? JAAPA. 2017 May;30(5):12- 15. DOI: 10.1097/01. JAA.0000515554.91731.82.

*Innate Immunity in Health and Disease*

6-[N(2-choro-4-fluorophenyl)

receptor 4-mediated cytokine production through suppression of intracellular signaling. Molecular Pharmacology April1,2006,69(4)1288- 1295. DOI:10.1124/mol.105.019695

Hazeki O, Kitazaki T, and Lizawa Y. A novel cyclohexene derivative, ethyl(6R)- assays. BioMed Research International 2017,2017:9176937,23pages. DOI:

[34] Savva A, and Roger T. Targeting toll-like receptors: promising therapeutic strategies for the management of sepsis-associated pathology and infectious diseases. Front

[35] Spirig R, Djafarzadeh S, Regueira T, Shaw SG, von Garnier C, et al. Effects of TLR Agonists on the Hypoxia-Regulated

Immunol. 2013 Nov 18;4:387. DOI:10.3389/fimmu.2013.00387

Transcription Factor HIF-1α and Dendritic Cell Maturation under Normoxic Conditions. PLOS ONE 2010 5(6): e10983. DOI: 10.1371/journal.

[36] Wood, E.G., Macdougall, C.E., Blythe, H. et al. HIF1α activation in dendritic cells under sterile conditions promotes an anti-inflammatory phenotype through accumulation of intracellular lipids. Sci Rep **10,** 20825

pone.0010983

(2020). DOI:10.1038/ s41598-020-77793-6

[37] Pandey S, Agrawal DK.

1 alpha in Toll-like receptor

Cell Res 19, 973-983 (2009). DOI:10.1038/cr.2009.44

[39] A Licata, MG Minissale, V Calvaruso, A Craxì A focus on epidemiology of drug-induced liver injury: analysis of a prospective cohort. Eur Rev Med Pharmacol Sci 2017,Vol. 21 - N. 1 Suppl, Pages: 112-121. Available from: https://www.europeanreview.org/

article/12449

Immunobiology of Toll-like receptors: emerging trends. Immunol Cell Biol. 2006 Aug;84(4):333-41. DOI: 10.1111/j.1440-1711.2006.01444.x.

[38] Nicholas S, and Sumbayev, V. The involvement of hypoxia-inducible factor

7/8-mediated inflammatory response.

[40] Luu B, Rodway GW. Does low-dose methotrexate deserve more respect from

10.1155/2017/9176937

sulfamoyl]cyclohex-1-ene-1-carboxylate (TAK-242), selectively inhibits toll-like

[28] Yee J, and Orchard D. Monitoring recommendations for oral azathioprine, methotrexate and cyclosporine in a pediatric dermatology clinic and

literature review. Australasian Journal of Dermatology 2018Feb,59(1):31-40. DOI:

[29] Wan S, Xiang Y, Fang W, and Huang D. The effect of methotrexate in combination with mifepristone on ectopic pregnancy: A meta-analysis. International Journal of Clinical and

2016,9(8):14990-15003. Available from:http://www.ijcem.com/files/

[30] Coskun M, Steenholdt C, de Boer NK and Nielsen OH. Pharmacology and

[31] Kaplowitz N. (2004) Drug induced liver injury. Clinical Infectious disease

[32] Bianchi G, Caporali R, Todoerti M, and Mattana P. Methotrexate and rheumatoid arthritis: Current evidence regarding subcutaneous versus oral routes of administration. ADVANCES IN THERAPY 2016;33:369-378. DOI:

[33] Roth AD and Lee MY. Idiosyncratic drug-induced liver injury (IDILI): Potential mechanisms and protective

optimization of thiopurines and methotrexate in inflammatory bowel disease. Clinical pharmacokinetics (2016)55,257-74. DOI: 10.1007/

2004Mar:38(Suppl2):S44-8.

10.1007/s12325-016-0295-8

10.1111/ajd.12526

Experimental Medicine

ijcem0028751.pdf

s40262-015-0316-9

DOI:10.1086/38446

**152**

[41] Campbell JM, Bateman E, Stephenson MD, Bowen JM, Keefe DM, and Peters MD. Methotrexate-induced toxicity pharmacogenetics: An umbrella review of systematic reviews and meta analysis. Cancer Chemotherapy and Pharmacology 2016 Jul;78(1):27-39. DOI: 10.1007/s00280-016-3043-5

[42] Singh D, Cho WC, and Upadhyay G. Drug-induced liver toxicity and prevention by herbal antioxidants: An overview. Frontiers In Physiology 1.6:363. DOI: 10.3389/ fphys.2015-00363

[43] Olayinka ET, Ore A, Adeyemo OA, and Ola OS. Ameliorative effect of gallic acid on methotrexate-induced hepatotoxicity in rat. Journal of xenobiotics 2016;6:6092. DOI: doi. org/10.408/xeno.2016,6092

[44] Fagone P, Mangano K, Mammana S, Pesce A, Pesce A, Caltabino R and Nicoletti F, et al. Identification of novel targets for the diagnosis and treatment of liver fibrosis. International Journal of Molecular Medicine 36:747-752,2015. DOI: 10.3892/ijmm.2015.2264

[45] Zhang N, Liang H, Farese RV, Li J, Musi N, Hussey SE. Pharmacological TLR 4 inhibition protects against acute and chronic fat-induced insulin resistance in rats. Plos ONE 10(7):e013575. DOI: 10.1371/journal. pone.013575

[46] Privitera G, Agarwal B, and Jalan R. Acute Liver Failure: Pathophysiologic basis, and the current and emerging therapies. EMJ Hepatol. 2014;1:99-107. Available from: https:// emj.emg-health.com/wp-content/ uploads/sites/2/2018/02/Acute-Liver-Failure-Pathophysiologic-Basis-And-The-Current-And-Emerging-Therapies.pdf

[47] Cao L, Quan XB, Zeng WJ, Yang XO, Wang MJ. Mechanisms of hepatocyte apoptosis. Journal of cell death 2016:919-26. DOI:10.4137/JCD.S39824

**155**

**Chapter 6**

**Abstract**

Human Herpetic Viruses and

*Majid Mohammed Mahmood and Murtada Hafedh Hussein*

Herpesviruses are large, spherical, enveloped viral particles with linear doublestranded DNA genome. Herpesvirus virion consists of an icosahedral capsid containing viral DNA, surrounded by a protein layer called tegument, and enclosed by an envelope consisting of a lipid bilayer with various glycoproteins. Herpesviruses persist lifelong in their hosts after primary infection by establishing a latent infection interrupted recurrently by reactivations. The Herpesviridae family is divided into three subfamilies; α-herpesviruses, β-herpesviruses, and γ-herpesviruses based on the genome organization, sequence homology, and biological properties. There are eight human herpes viruses: Herpes simplex virus type 1 and 2 (HSV-1, −2) and-Varicella-zoster virus (VZV), which belong to the α-herpesvirus subfamily; Human cytomegalovirus (HCMV), and Human herpesvirus type 6 and 7 (HHV-6,HHV-7), which belong to the β-herpesvirus subfamily; and Epstein–Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) or Human herpesvirus 8 (HHV-8), which belong to the γ-herpesvirus subfamily. Within this chapter, we summarize the current knowledge about EBV and CMV, regarding their genome organization, structural characteristics, mehanisms of latency, types of infections, mechanisms of immune escape and prevention. Epstein–Barr Virus (EBV) genome encodes over 100 proteins, of which only (30) proteins are well characterized, including the proteins expressed during latent infection and lytic cycle proteins. Based on major variation in the EBNA-2 gene sequence, two types of EBV are recognized, EBV type 1 and 2. Epstein–Barr virus types occur worldwide and differ in their geographic distribution depending on the type of virus. **EBV** spreads most commonly through bodily fluids, especially saliva. However, EBV can also spread through blood, blood transfusions, and organ transplantations. The EBV is associated with many malignant diseases such as lymphomas, carcinomas, and also more benign such as infectious mononucleosis, chronic active infection. The EBV has also been suggested as a trigger/ cofactor for some autoimmune diseases. Overall, 1–1.5% of the cancer burden worldwide is estimated to be attributable to EBV The latently infected human cancer cells express the most powerful monogenic proteins, LMP-1 and LMP-2(Latent Membrane Protein-1,-2), as well as Epstein–Barr Nuclear Antigens (EBNA) and two small RNAs called Epstein–Barr Encoded Small RNAs (EBERs). The EBV can evade the immune system by its gene products that interfering with both innate and adaptive immunity, these include EBV-encoded proteins as well as small noncoding RNAs with immune-evasive properties. Currently no vaccine is available, although there are few candidates under evaluation. Human cytomegalovirus (HCMV) is a ubiquitous beta herpesvirus type 5 with seroprevalence ranges between 60 to 100% in developing countries. CMV is spread from one person to another, usually by direct

Immune Profiles

*Marwa Mohammed Ali Jassim,* 

#### **Chapter 6**

## Human Herpetic Viruses and Immune Profiles

*Marwa Mohammed Ali Jassim, Majid Mohammed Mahmood and Murtada Hafedh Hussein*

#### **Abstract**

Herpesviruses are large, spherical, enveloped viral particles with linear doublestranded DNA genome. Herpesvirus virion consists of an icosahedral capsid containing viral DNA, surrounded by a protein layer called tegument, and enclosed by an envelope consisting of a lipid bilayer with various glycoproteins. Herpesviruses persist lifelong in their hosts after primary infection by establishing a latent infection interrupted recurrently by reactivations. The Herpesviridae family is divided into three subfamilies; α-herpesviruses, β-herpesviruses, and γ-herpesviruses based on the genome organization, sequence homology, and biological properties. There are eight human herpes viruses: Herpes simplex virus type 1 and 2 (HSV-1, −2) and-Varicella-zoster virus (VZV), which belong to the α-herpesvirus subfamily; Human cytomegalovirus (HCMV), and Human herpesvirus type 6 and 7 (HHV-6,HHV-7), which belong to the β-herpesvirus subfamily; and Epstein–Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) or Human herpesvirus 8 (HHV-8), which belong to the γ-herpesvirus subfamily. Within this chapter, we summarize the current knowledge about EBV and CMV, regarding their genome organization, structural characteristics, mehanisms of latency, types of infections, mechanisms of immune escape and prevention. Epstein–Barr Virus (EBV) genome encodes over 100 proteins, of which only (30) proteins are well characterized, including the proteins expressed during latent infection and lytic cycle proteins. Based on major variation in the EBNA-2 gene sequence, two types of EBV are recognized, EBV type 1 and 2. Epstein–Barr virus types occur worldwide and differ in their geographic distribution depending on the type of virus. **EBV** spreads most commonly through bodily fluids, especially saliva. However, EBV can also spread through blood, blood transfusions, and organ transplantations. The EBV is associated with many malignant diseases such as lymphomas, carcinomas, and also more benign such as infectious mononucleosis, chronic active infection. The EBV has also been suggested as a trigger/ cofactor for some autoimmune diseases. Overall, 1–1.5% of the cancer burden worldwide is estimated to be attributable to EBV The latently infected human cancer cells express the most powerful monogenic proteins, LMP-1 and LMP-2(Latent Membrane Protein-1,-2), as well as Epstein–Barr Nuclear Antigens (EBNA) and two small RNAs called Epstein–Barr Encoded Small RNAs (EBERs). The EBV can evade the immune system by its gene products that interfering with both innate and adaptive immunity, these include EBV-encoded proteins as well as small noncoding RNAs with immune-evasive properties. Currently no vaccine is available, although there are few candidates under evaluation. Human cytomegalovirus (HCMV) is a ubiquitous beta herpesvirus type 5 with seroprevalence ranges between 60 to 100% in developing countries. CMV is spread from one person to another, usually by direct and prolonged contact with bodily fluids, mainly saliva, but it can be transmitted by genital secretions, blood transfusion and organ transplantation. In addition, CMV can be transmitted vertically from mother to child. CMV infection can result in severe disease for babies, people who receive solid organ transplants or bone marrow/stem cell transplants and people with severe immune suppression such as advanced human immunodeficiency virus (HIV) infection. The HCMV has several mechanisms of immune system evasion. It interferes with the initiation of adaptive immune responses, as well as prevent CD8+ and CD4+ T cell recognition interfering with the normal cellular MHC Class I and MHC Class II processing and presentation pathways. Challenges in developing a vaccine include adeptness of CMV in evading the immune system. Though several vaccine candidates are under investigation.

**Keywords:** human cytomegalovirus, Epstein-Barr virus, mononucleosis, transplantation, immune evasion, oncogenesis

#### **1. Introduction**

#### **1.1 The Herpesviridae family**

Herpesviridae is a large family of double-stranded DNA viruses, which is included in the recently classified order Herpesvirales. This family can be further classified into three distinct subfamilies: *Alphaherpesvirinae, Betaherpesvirinae, and Gammaherpesvirinae*, according to their biology and DNA genomic sequence [1]. The *Alphaherpesvirinae* subfamily includes five distinct genera, *Simplexvirus* and *Varicellovirus* are most important members causes infection to human. The members of Herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) infect almost 85% of the world human population and cause orolabial.herpes and genital herpes; other members of the family include varicella-zoster virus (VZV), which is responsible for chicken-pox and shingles. The *Betaherpesvirinae* subfamily includes four genera, the most important members that infect the human are Human *cytomegalovirus* also known as human herpesvirus 5 (HCMV or HHV-5) and Human herpesviruses 6 and 7 (HHV-6 and HHV-7) [2].

The *Gammaherpesvirinae* subfamily is composed of four distinct genera, *Lymphocryptovirus* (LCV) and *Rhadinovirus* (RDV)infect the human, Taxonomically, the oncogenic Epstein–Barr virus (EBV) is also designated as human herpesvirus 4 (HHV-4) belongs to the genus lymphocryptovirus (LCV) and it is the only human pathogen of this genus. The RDV Kaposi's sarcoma-associated virus (KSHV, also known as HHV-8), another oncogenic herpesvirus, is the only known human RDV [3]. The *Gammaherpesviruses* may promote oncogenic effects and also contribute to the development of malignancies but this is a rare outcome [2]. Altogether herpesviruses can establish latent infection within specific tissues, with immune surveillance evasion. The human herpesviruses and their diseases are summarized in (**Table 1**).

#### **1.2 Epstein–Barr Virus (EBV)**

EBV is ubiquitous virus, with a seroprevalence of more than 90% of the adult population worldwide. It was first identified in 1964 by Anthony Epstein's group in a cell line from a Burkitt's lymphoma biopsy [4, 5]. The EBV has also been identified as a B lymphotropic oncogenic virus owing to its capacity to convert resting B lymphocytes in vitro, inducing continuous dissemination of infected B cells and producing lymphoblastic cell lines (LCLs) [6]. This discovery was central to the identification of EBV as the first nominee human tumor virus. Subsequently,

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**Table 1.**

perfectly immune-controlled [2].

*Taxonomy of Human Herpesviruses [3].*

*1.2.1 The EBV Virion and Genome organization*

*Human Herpetic Viruses and Immune Profiles DOI: http://dx.doi.org/10.5772/intechopen.96340*

**Subfamily Genus Species Disease**

*Varicellovirus Human alphaherpesvirus* 3

*Roseolovirus Human betaherpesvirus* 7

6A, 6B

6B)

*Rhadinovirus Human gammaherpesvirus* 8

(Herpes simplex virus 1) *Human alphaherpesvirus* 2 (Herpes simplex virus 2)

(*Varicella Zoster* virus)

(Human cytomegalovirus)

(Human herpesvirus 7)

*Human betaherpesvirus*

(Epstein–Barr Virus)

(Kaposi's sarcomaassociated virus or Human

herpesvirus 8)

(Human herpesvirus 6A,

Acute Herpetic gingivostomatitis, Keratitis, Conjunctivitis, Encephalitis, Dermal whitlow, Herpes labialis Herpes genitalis

Chickenpox/ shingles

Congenital abnormalities

subitum

Infectious mononucleosis (Glandular fever), Burkitt's lymphoma, Hodgkin's lymphoma, Nasopharyngeal carcinoma, Oral hairy leukoplakia

Kaposi's sarcoma

Febrile illnesses

Infant rash Exanthem

*Alphaherpesvirinae Simplexvirus Human alphaherpesvirus* 1

*Betaherpesvirinae Cytomegalovirus Human betaherpesvirus* 5

*Gammaherpesvirinae Lymphocryptovirus Human gammaherpesvirus* 4

EBV was correlated with a variety of clinical malignancies, including Hodgkin's Lymphoma (HL), post-transplant lymphoproliferative disease (PTLD) and X-linked lymphoproliferative disease (XLPD). The potential to invade other cell types other than B lymphocyte, such as T, natural killer (NK) and epithelial cells, has led to the association of EBV with other malignancies: peripheral T cell, nasal T or NK cell lymphomas, gastric and nasopharyngeal carcinomas (NPC) [2, 7]. However, infection with EBV induces contagious mononucleosis during or after adolescence [8]. Even though EBV exhibits a strong growth transforming capacity, that asymptomatically infects up to 95% of the human population, whereas it is

The virus is 122–180 nm in diameter. Epstein–Barr virion contains a linear, double-stranded DNA genome wrapped on an icosahedral capsid, approximately (100–110) nm in diameter, containing 162 capsomeres with a pore running down the long axis. The protein tegument with viral and cellular proteins including actin, tubulin, and cofilin separates the nucleocapsid from the lipid envelope that coats the virus and contains numerous viral glycoproteins (GP) spikes such as gp350/220, gp42, GH, GB and gp150 on the outer surface. These glycoproteins play an important role in cell tropism and recognition of receptors [8, 9] as shown in the **Figure 1**.

*Human Herpetic Viruses and Immune Profiles DOI: http://dx.doi.org/10.5772/intechopen.96340*


#### **Table 1.**

*Taxonomy of Human Herpesviruses [3].*

EBV was correlated with a variety of clinical malignancies, including Hodgkin's Lymphoma (HL), post-transplant lymphoproliferative disease (PTLD) and X-linked lymphoproliferative disease (XLPD). The potential to invade other cell types other than B lymphocyte, such as T, natural killer (NK) and epithelial cells, has led to the association of EBV with other malignancies: peripheral T cell, nasal T or NK cell lymphomas, gastric and nasopharyngeal carcinomas (NPC) [2, 7]. However, infection with EBV induces contagious mononucleosis during or after adolescence [8]. Even though EBV exhibits a strong growth transforming capacity, that asymptomatically infects up to 95% of the human population, whereas it is perfectly immune-controlled [2].

#### *1.2.1 The EBV Virion and Genome organization*

The virus is 122–180 nm in diameter. Epstein–Barr virion contains a linear, double-stranded DNA genome wrapped on an icosahedral capsid, approximately (100–110) nm in diameter, containing 162 capsomeres with a pore running down the long axis. The protein tegument with viral and cellular proteins including actin, tubulin, and cofilin separates the nucleocapsid from the lipid envelope that coats the virus and contains numerous viral glycoproteins (GP) spikes such as gp350/220, gp42, GH, GB and gp150 on the outer surface. These glycoproteins play an important role in cell tropism and recognition of receptors [8, 9] as shown in the **Figure 1**.

**Figure 1.** *The structure of EBV viral particle.*

The double-stranded DNA (172Kb) linear genome encodes more than 100 proteins as well as non-coding functional RNAs (EBER RNAs, BART miRNAs, and BHRF1 miRNAs). There are some similar tandem terminal repeats (TR) of 0.5 kb at each terminal of the genome [10] and other internal direct repeats of 3 kb (IR) including the latency promoter (Wp) and the special short unique sequence domains (US)and UL (long). The US and UL sequences comprise nearly all of the genome encoding capacity [11] as shown in **Figure 2**. The EBV genome is classified as C genome, which is linear in a virus particle, but distributed as an episome in the nucleus of infected cells; circulating occurs by terminal repeat units (TRs) following B cell infection with EBV [11]. The first cloned and sequenced EBV strain was typing 1 EBV: B95.8, this strain was obtained from an infectious mononucleosis patient's. Sequencing was based on previously generated EcoRI and BamHI restriction fragments (**Figure 3**). B95.8 strain is commonly used in labs around the world; however, a 13.6 kb portion of its genome is incomplete. Subsequently, the missing fragment was sequenced from the Raji strain and a revised EBV consensus genome was released several years later [12].

#### *1.2.2 The EBV classification*

Two major types of EBV, type 1 and 2, have been described in humans based on major variations in EBNA-2 gene sequence [11]. Type 1 is dominant throughout most of the world, but the two types are equally prevalent in Africa. The EBNA-2 is the most variable locus in the EBV genome which is characterized by 70% identity at the level of nucleotide sequence whereas only 56% similarity at the amino acid level between these two types (3). In addition, the variation between type 1 and type 2 is also linked to the sequence variation in the viral latent genes EBNA-3A, EBNA-3B, EBNA-3C and EBNA-LP [13].

#### *1.2.3 Epstein Barr virus life cycle*

#### *1.2.3.1 Cell attachment and viral entry*

The initial attachment of EBV is mainly regulated by the association between its envelope protein (gp350/220) and the cellular complement component receptor 2 (CR2/CD21) protein located on the B cell surface. This association activates Cluster of differentiation (CD21) receptor aggregation in the plasma membrane and also a tyrosine kinase signal transduction through CD19 that contributes to Nuclear factor-kappa B (NF- kB) activation and cell cycle entry [14].

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*Human Herpetic Viruses and Immune Profiles DOI: http://dx.doi.org/10.5772/intechopen.96340*

*Linear Organization of the EBV genome.*

*Circular Organization of the EBV genome.*

**Figure 2.**

**Figure 3.**

The attachment of the second viral glycoprotein gp42 to the human leukocyte antigen class II receptor (HLA class II) activates the viral envelope fusion with the membrane of the cell and the viral entrance in a cycle that relies on the glycoprotein complex GH/GL and also on GB [15]. The GH/GL complex is supposed to serve as a receptor that activates GB-mediated fusion after gp42 binding to HLA Class II molecules. Thereafter, virion nucleocapsids are released into the cytoplasm and transported to nuclear pores on microtubules [13]. As a result, the viral linear genome is transferred to the nucleus of B lymphocyte and the viral genome is then retained in the nucleus as a covalently locked extrachromosomal episome [16].

For epithelial cells, as there are no CD21 or HLA class II molecules on their surface, the entrance of EBV does not involve gp350/220 and gp42. Viral BMRF2 protein can mediates interaction with cellular β1 integrins [14]. The fusion of viral envelope is activated by the attachment of the viral gH/gL complex to 5–007vβ6/8 integrins, which is confirmed by the effectiveness of infection in virions missing gp350/220 glycoproteins. The EBV virion expresses three- gH / gL/ gp42 and two- gH/gL glycoprotein complexes that grant the capacity to invade either B cells or epithelial cells [13]. The virus is endocytosed into a low pH vesicle where fusion occurs after the interplay of EBV glycoprotein gp350 and receptor type 2 (CR2). Glycoprotein gp42 is bound directly to GH and transforms dimeric GHGL in a trimeric gHgLgp42, modifying the conformation of gp42 to cause its attachment to the human leukocyte antigen (HLA) class II molecule. It will allow the central fusion machine to support effective B cell infection. Besides, GH can bind cellular components [15].

*Human Herpetic Viruses and Immune Profiles DOI: http://dx.doi.org/10.5772/intechopen.96340*

**Figure 2.**

*Linear Organization of the EBV genome.*

**Figure 3.** *Circular Organization of the EBV genome.*

The attachment of the second viral glycoprotein gp42 to the human leukocyte antigen class II receptor (HLA class II) activates the viral envelope fusion with the membrane of the cell and the viral entrance in a cycle that relies on the glycoprotein complex GH/GL and also on GB [15]. The GH/GL complex is supposed to serve as a receptor that activates GB-mediated fusion after gp42 binding to HLA Class II molecules. Thereafter, virion nucleocapsids are released into the cytoplasm and transported to nuclear pores on microtubules [13]. As a result, the viral linear genome is transferred to the nucleus of B lymphocyte and the viral genome is then retained in the nucleus as a covalently locked extrachromosomal episome [16].

For epithelial cells, as there are no CD21 or HLA class II molecules on their surface, the entrance of EBV does not involve gp350/220 and gp42. Viral BMRF2 protein can mediates interaction with cellular β1 integrins [14]. The fusion of viral envelope is activated by the attachment of the viral gH/gL complex to 5–007vβ6/8 integrins, which is confirmed by the effectiveness of infection in virions missing gp350/220 glycoproteins. The EBV virion expresses three- gH / gL/ gp42 and two- gH/gL glycoprotein complexes that grant the capacity to invade either B cells or epithelial cells [13].

The virus is endocytosed into a low pH vesicle where fusion occurs after the interplay of EBV glycoprotein gp350 and receptor type 2 (CR2). Glycoprotein gp42 is bound directly to GH and transforms dimeric GHGL in a trimeric gHgLgp42, modifying the conformation of gp42 to cause its attachment to the human leukocyte antigen (HLA) class II molecule. It will allow the central fusion machine to support effective B cell infection. Besides, GH can bind cellular components [15].

The epithelial cells do not constitutively express HLA class II, which makes gp42 useless in the process of fusion. The interaction of dimeric GHGL complexes with integrins, however, replaces the cell fusion caused by the interaction between gp42 and HLA class II. The use of dimeric GHGL complexes to cause epithelial cell fusion and gHgLgp42 trimeric complexes to contribute to B cell fusion was expected that the virus would trigger B cells and epithelial cells to alter the viral tropism: The gp42 spike in epithelial viral particles makes it 100 times more infectious than the virus produced from B-cells. The opposite is not so dramatic: the B-virus is five times more contagious for the epithelial cell than the epithelial virus [17]. After binding to the primary B cell, most virions do not internalize with the epithelial cell and the infection can be significantly increased by co-culturing with EBV negative B cells. Such virions stay on the surface of the cell B and can then be passed via the formation of the intracellular synapse to CR2-negative epithelial cells. This transfer technique involves the interaction between gp350-CR2 and GH and GB viral glycoproteins. This mechanism has been suggested to allow EBV to enter both lymphoid and epithelial cells simultaneously [18].

#### *1.2.3.2 EBV Lytic Infection*

The lytic infection is characterized by the active release of new contagious virus particles, either infecting new human hosts or infecting other naive B cells in the same host. The lytic cycle is divided into three stages: Immediate-early (IE), Early (E), and Late (L). The expression of immediate early BZLF1 and BRLF1 genes included in the activation of the lytic process is activated by signal transduction by the B cell receptor (BCR) [15]. The BZLF1 is a viral transactivator protein responsible for activating the production of lytic genes and the repression of latent genes, resulting in cells' death and the release of contagious virons. The signal transduction of BCR initiates BRLF1 development and also improves its production allowing the transition from latency to lytic cycle [14]. The BZLF1 protein is a bZIP-specific transcription factor close to c-FOS and C/EBP. The BZFL1 and BRLF1 motivate functions of early genes, such as viral DNA polymerase (BALF5) and thymidine kinase, to initialize viral DNA replication from the lytic origin of replication (OriLyt) in tandem with other direct and early gene products [13]. Late lytic genes encode viral structural proteins, including tegument proteins, glycoproteins, and BcLF1 main capsid proteins. Newly synthesized viral DNAs are packed into nucleocapsids in the nucleus of the cell, which moves across the nuclear membrane to the cytoplasm, creating vesicles carrying virons with an envelope. The vesicles fuse with the plasma cell membrane and the virus particles exocytose [19].

#### *1.2.3.3 Latency*

Herpesviruses are distinguished by their ability to establish and sustain a latent infection in their hosts. Latent EBV expresses its genes in one of three latency systems: Latency I, II, or III variations in either of these systems assist in the development of a distinct series of viral RNAs and proteins [20] **Table 2** and **Figure 4**. This chronic infection is characterized by inhibition of viral replication and viral dormancy, and immune evasion in the host. The EBV determines latency in the B cell pool which is the long-term reservoir for the virus in vivo*.* Naive B cells infected with EBV in the Waldeyer ring proliferate as activated B bursts, which are close to antigen-activated B lymphocytes in terms of the structure and morphology of their cell surface [24].

Opposite to the lytic infection, replication of the viral genome in latent infection occurs through host DNA polymerase and from a separate source, Orig of

**161**

**Table 2.**

latently infected B cells [26].

*Human Herpetic Viruses and Immune Profiles DOI: http://dx.doi.org/10.5772/intechopen.96340*

**EBV latent protein Function related to B-cell lymphomagenesis**

cell transformation

EBAN3B Virus-encoded tumor suppressor protein

cell transformation

blocking cellular apoptosis

regulation LMP2B Regulates LMP2A functions

Essential for B cell transformation

EBNA1 Regulation of viral DNA replication and transcription of many viral and cellular

EBNA2 One of the main viral transcription factors; In combination with EBNALP, EBNA2

EBNALP EBNA2-mediated transcription activator, both for viral and cellular genes; Bypassing

EBNA3A Together with EBNA3C, it suppresses the genetic transcription of BIM, p14, p15, p16

EBNA3C Together with EBNA3A, it suppresses the genetic transcription of BIM, p14, p15, p16

LMP1 Functionally mimics CD40 signaling pathway; one of the major transcriptional

LMP2A Functionally mimics BCR signaling pathway; prevents apoptosis; EBV latency

activated dsRNA dependent protein kinase (PKR)

EBERs Most of the non-coding viral RNA is found in all forms of latency programs; Affects

miRNAs Transcribed from BART and BHRF1 loci; maintains latently infected B cells through

apoptosis; Activates autophagy necessary for B cell transformation

genes; It facilitates p53 disintegration and thus promotes tumorigenesis

regulates the transcription of many of viral and cellular genes; Fundamental for B

and p18 through epigenetic regulation; Prevents differentiation of B cells into plasma;

and p18 through epigenetic regulation; Assists G1-S and G2-M transformations of the cell cycle; Ubiquitin-proteasome pathway; Supresses p53-, E3F1- and Bim-mediated

regulators; Mainly activates NF-kB, JAK/STAT, ERK MAPK, IRF and Wn't signaling pathways; Induces BCL-2 and a20 expression to prevent apoptosis; Essential for B

the innate immune response and gene expression; Inhibits apoptosis dependent on

the innate immune response of cells; Fundamental for B cell transformation

replication. During latent infection, the viral genome is present as a closed circular, extrachromosomal plasmid or episome. The viral DNA is wrapped with host histone molecules and replicates steadily once throughout the cell cycle together with the host genome [25], this enabled EBV infected B blasts during proliferation to express all latent EBV genes which are known as latency III or growth-program

*Impact of latent antigens in EBV on B-cell transformation and subsequent development of lymphoma [21–23].*

This is achieved by the expression of two viral latent membrane proteins (LMPs), LMP-1, and LMP-2A, which constitute a functional homolog of the CD40 receptor in B lymphocytes and often mimic the constitutively active BCR, respectively [26]. B cell migrates to nearby primary follicles to form germ centers and the viral transcription system switches to latency II or a default system to enable the B cells to differentiate into memory B cells, Latency II is characterized by the expression of LMPs and EBNA-1 protein.. In the absence of antigen-mediated signals, LMPs are necessary to provide cell survival signals needed to prevent apoptosis of

Epstein Barr-virus nuclear antigen-1(EBNA-1) protein is important for EBV DNA replication and for preservation of viral genome in the cells [20]. The memory B cells lately infected reach peripheral circulation and represent viral persistence reservoir; [27]. Such latently infected memory B cells with EBV are distinguished by a silence of the expression of viral protein in a program called latency 0 or latency-program which is intended to permit immune evasion and therefore lifelong

that play important role in cell activation and proliferation.

*Human Herpetic Viruses and Immune Profiles DOI: http://dx.doi.org/10.5772/intechopen.96340*


#### **Table 2.**

*Impact of latent antigens in EBV on B-cell transformation and subsequent development of lymphoma [21–23].*

replication. During latent infection, the viral genome is present as a closed circular, extrachromosomal plasmid or episome. The viral DNA is wrapped with host histone molecules and replicates steadily once throughout the cell cycle together with the host genome [25], this enabled EBV infected B blasts during proliferation to express all latent EBV genes which are known as latency III or growth-program that play important role in cell activation and proliferation.

This is achieved by the expression of two viral latent membrane proteins (LMPs), LMP-1, and LMP-2A, which constitute a functional homolog of the CD40 receptor in B lymphocytes and often mimic the constitutively active BCR, respectively [26]. B cell migrates to nearby primary follicles to form germ centers and the viral transcription system switches to latency II or a default system to enable the B cells to differentiate into memory B cells, Latency II is characterized by the expression of LMPs and EBNA-1 protein.. In the absence of antigen-mediated signals, LMPs are necessary to provide cell survival signals needed to prevent apoptosis of latently infected B cells [26].

Epstein Barr-virus nuclear antigen-1(EBNA-1) protein is important for EBV DNA replication and for preservation of viral genome in the cells [20]. The memory B cells lately infected reach peripheral circulation and represent viral persistence reservoir; [27]. Such latently infected memory B cells with EBV are distinguished by a silence of the expression of viral protein in a program called latency 0 or latency-program which is intended to permit immune evasion and therefore lifelong

#### **Figure 4.**

#### *Model for the establishment of EBV persistent latent infection.*

persistence on the host. The expression EBNA-1 is enabled and allows the division of the viral genome in the cells carrying the virus. This is known as the transcription program Latency I or EBNA-1 only program [26]. From peripheral circulation, latently infected memory B cells migrate into oropharynx and tonsils and then differentiate into plasma antibody-producing cells. Reactivation of the virus is triggered and infectious viruses are created as they bear the virus. Therefore, these viral particles will infect additional hosts with new naive B cells [27].

#### *1.2.4 Transmission*

The oral route is the primary route of the EBV transmission commonly through bodily fluids, especially saliva [28]. However, it has been reported that EBV infection can also be transmitted after the transfusion of a large volume of fresh blood [29]. Although EBV has been detected in cervical secretions of 8% -28%, of

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*1.2.5 EBV epidemiology*

*1.2.6 EBV clinical features*

attributable to EBV [3].

*1.2.6.1 Primary EBV infection*

syndrome, encephalitis, and meningitis [35].

women, it is still controversy on whether EBV is transmitted through sexual contact [30]. Possible spread via organ transplantation can occur which is of particular concern in association with subsequent infection by EBV [25]. Transmission by milk is also a possible route, but is a non-significant mode of EBV transmission [31].

Epstein–Barr virus types occur worldwide, but they differ in their geographic distribution. For instance, Type 1 is prevalent in population from Europe, America, China, and South Asia, while Type 2 is less prevalent in these populations and is more observed in African and Papua New Guinean populations. Over 90–100% of adults have been infected with EBV, and the infection is most commonly affecting those patients aged 2 to 4 years and those aged 15 years. Epstein–Barr virus causes approximately 90% of the cases of infectious mononucleosis, which is commonly seen in both the community and the hospital setting. Among infants and young children who are primarily infected with EBV, in Africa, and where Burkitt lymphoma is common, 50% of them are infected with this virus before their 1 year of age. About 70% of cases of PTLD are associated with Epstein–Barr virus (EBV), especially in cases that occur early after transplantation [32]. Recent studies from Kenya reported a striking overlap between increased incidence of malaria transmission and Burkitt lymphoma [33]. Furthermore, various studies have demonstrated the presence of 8: 14 translocation in both the endemic African Burkitt lymphoma

and in the non-endemic tumor type (Europe, America, and Japan).

The EBV is associated with many malignant diseases such as lymphomas, carcinomas, and also more benign such as infectious mononucleosis, chronic active infection. The EBV has also been suggested as a trigger/cofactor for some autoimmune diseases. Overall, 1–1.5% of the cancer burden worldwide is estimated to be

The primary EBV infections of infants and children are often asymptomatic or have nonspecific symptoms, but infections of adolescents and adults frequently result in infectious mononucleosis (IM). Around 80% of infected adults mostly experience symptoms, including sore throat, cervical lymphadenopathy, weakness, upper respiratory infection, headache, reduced appetite, fever, and myalgia (muscle aches). It is characterized by a large number of lymphocytes, mainly CD8 + T-cells, which, as opposed to healthy individuals, can reach five to ten times more numbers in the blood. The causes of this expansion of T-cells in IM are not clear, but factors such as failure of natural immune control by natural killer (NK) cell, memory CD8+

T cells of memory of EBV or genetic background have been suggested [34].

The severity of symptoms in primary EBV infection is associated with age and immune system of the patients. The complications of the disease include splenomegaly, and/or chronic hepatitis, pneumonia and lymphadenitis. Less common are complications, such as hemolytic/aplastic anemia, myocarditis, Guillain–Barré

Chronic active EBV infection is a rare disorder characterized by the presence of severe illness of more than six months' duration, high virus-specific antibody titers and organ disease with the demonstration of EBV antigens or EBV DNA in tissue [35]. women, it is still controversy on whether EBV is transmitted through sexual contact [30]. Possible spread via organ transplantation can occur which is of particular concern in association with subsequent infection by EBV [25]. Transmission by milk is also a possible route, but is a non-significant mode of EBV transmission [31].

#### *1.2.5 EBV epidemiology*

Epstein–Barr virus types occur worldwide, but they differ in their geographic distribution. For instance, Type 1 is prevalent in population from Europe, America, China, and South Asia, while Type 2 is less prevalent in these populations and is more observed in African and Papua New Guinean populations. Over 90–100% of adults have been infected with EBV, and the infection is most commonly affecting those patients aged 2 to 4 years and those aged 15 years. Epstein–Barr virus causes approximately 90% of the cases of infectious mononucleosis, which is commonly seen in both the community and the hospital setting. Among infants and young children who are primarily infected with EBV, in Africa, and where Burkitt lymphoma is common, 50% of them are infected with this virus before their 1 year of age. About 70% of cases of PTLD are associated with Epstein–Barr virus (EBV), especially in cases that occur early after transplantation [32]. Recent studies from Kenya reported a striking overlap between increased incidence of malaria transmission and Burkitt lymphoma [33]. Furthermore, various studies have demonstrated the presence of 8: 14 translocation in both the endemic African Burkitt lymphoma and in the non-endemic tumor type (Europe, America, and Japan).

#### *1.2.6 EBV clinical features*

The EBV is associated with many malignant diseases such as lymphomas, carcinomas, and also more benign such as infectious mononucleosis, chronic active infection. The EBV has also been suggested as a trigger/cofactor for some autoimmune diseases. Overall, 1–1.5% of the cancer burden worldwide is estimated to be attributable to EBV [3].

#### *1.2.6.1 Primary EBV infection*

The primary EBV infections of infants and children are often asymptomatic or have nonspecific symptoms, but infections of adolescents and adults frequently result in infectious mononucleosis (IM). Around 80% of infected adults mostly experience symptoms, including sore throat, cervical lymphadenopathy, weakness, upper respiratory infection, headache, reduced appetite, fever, and myalgia (muscle aches). It is characterized by a large number of lymphocytes, mainly CD8 + T-cells, which, as opposed to healthy individuals, can reach five to ten times more numbers in the blood. The causes of this expansion of T-cells in IM are not clear, but factors such as failure of natural immune control by natural killer (NK) cell, memory CD8+ T cells of memory of EBV or genetic background have been suggested [34].

The severity of symptoms in primary EBV infection is associated with age and immune system of the patients. The complications of the disease include splenomegaly, and/or chronic hepatitis, pneumonia and lymphadenitis. Less common are complications, such as hemolytic/aplastic anemia, myocarditis, Guillain–Barré syndrome, encephalitis, and meningitis [35].

Chronic active EBV infection is a rare disorder characterized by the presence of severe illness of more than six months' duration, high virus-specific antibody titers and organ disease with the demonstration of EBV antigens or EBV DNA in tissue [35].

#### *1.2.6.2 EBV reactivation and EBV associated diseases*

The reactivation of latent EBV infection has been shown to occur following impairment of the cellular immune response which is important in the long-term control of persistent EBV infection. Chronic uncontrolled EBV reactivation may result in the development of carcinoma. The followings are diseases and cancers associated with EBV infection [2, 36–39]:


About 90% of head and neck cancers are squamous cell carcinoma (SCC), they originate from the mucosal lining that, causes tumor development in the nasal cavity and mouth, nasopharynx, larynx, esophagus and paranasal sinuses [44]. The International Agency for Research on Cancer (IARC) estimated that 16% of total

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*1.2.6.3 C. Autoimmune diseases.*

new cancers, as well as 20% of deaths caused by cancers worldwide, were due to infections with EBV [2, 7]. Sinonasal carcinoma is a rare tumor comprised of about

EBV has variously been linked with a number of autoimmune diseases including multiple sclerosis (MS), systemic lupus erythematosus (SLE), Sjögren's syndrome (SS) and rheumatoid arthritis (RA). The EBV links with these diseases include raised titers of EBV antibodies, decreased T cell response to EBV and elevated EBV viral load. It has been suggested that EBV triggers the activation state of the immune system by inducing the development of pro-inflammatory mediators,

Oncogenesis is a cytological, genetic, and cellular transformation process that results in malignant tumors. Discovery of viral oncogenes and the discovery that they are derived from cellular genes called protooncogenes led to the understanding that c-onc genes have roles in different tumor types. The activation of viral oncogenes requires genetic changes in cellular protooncogenesby 3 genetic mechanisms: (a) Mutation (b) Gene amplification (c) Chromosome rearrangements. These mechanisms result in either an increase in protooncogene expression or a change in protooncogene structure [47]. The EBV-mediated B-cell change is associated with a global improvement in viral and cellular expression of genes. The biologic characteristics of the virus were instantly fascinating, as it was shown that cell lines could be determined from samples of Burkitt's lymphoma (BL) and could propagate a virus that could strike primary B cells with EBV and turn them into immortalized cell lines [48]. This study of molecular phenotype led to the discovery of viral proteins that are necessary for latent infection and needed for cell transformation

EBV encoding oncogenes induce the changes in the host cellular signaling pathways that control proliferation, differentiation, cell death, genomic integrity,

LMP1, LMP2A, and LMP2B, latent membrane proteins are generated of the common viral locus with converging and interfering primary transcripts [50]. The LMP1 is one of the main EBV-encoded oncoproteins and it is a constitutively active mimic of cellular CD40 receptor. It is critically important for the EBV-induced B-cell transformation via the activation of NF-κB, c-Jun N-terminal kinase (JNK), and p38 cascades [21]. LMP1 also regulates cellular apoptosis by triggering the NF-κB pathway by increasing the antiapoptotic expression of Bcl2 via IRAK1 and TRAF6 whereIRAK1 is necessary for both p38 and p65/RelA phosphorylation [50]. Also, LMP1-stimulated proapoptotic polycomb complex protein (Bmi-1) is further been recruiting by EBNA3C for the transcriptional funnel of other genes. LMP2A acts as a functional homolog of the B-cell receptor (BCR) and thus promotes the survival of B-cells. Likewise, it is essential for the growth transformation of germinal center-derived B-cells which are BCR negative [21]. LMP2B negatively regulates LMP2A functions and transition from latent to lytic activation by depleting LMP2A-mediated BCR cross-linking and restoring Ca2+ mobilization [51].

1% of all cancers and 3% of all head and neck cancers [44, 45].

which may play a role in autoimmune pathogenesis [46].

*1.2.7 EBV oncogenesis and association of latency type*

[49]. The mechanisms of EBV oncogenesis include:

*1.2.7.1 Alteration of host cellular signaling pathways*

and recognition by the immune system.

#### *Human Herpetic Viruses and Immune Profiles DOI: http://dx.doi.org/10.5772/intechopen.96340*

new cancers, as well as 20% of deaths caused by cancers worldwide, were due to infections with EBV [2, 7]. Sinonasal carcinoma is a rare tumor comprised of about 1% of all cancers and 3% of all head and neck cancers [44, 45].

#### *1.2.6.3 C. Autoimmune diseases.*

EBV has variously been linked with a number of autoimmune diseases including multiple sclerosis (MS), systemic lupus erythematosus (SLE), Sjögren's syndrome (SS) and rheumatoid arthritis (RA). The EBV links with these diseases include raised titers of EBV antibodies, decreased T cell response to EBV and elevated EBV viral load. It has been suggested that EBV triggers the activation state of the immune system by inducing the development of pro-inflammatory mediators, which may play a role in autoimmune pathogenesis [46].

#### *1.2.7 EBV oncogenesis and association of latency type*

Oncogenesis is a cytological, genetic, and cellular transformation process that results in malignant tumors. Discovery of viral oncogenes and the discovery that they are derived from cellular genes called protooncogenes led to the understanding that c-onc genes have roles in different tumor types. The activation of viral oncogenes requires genetic changes in cellular protooncogenesby 3 genetic mechanisms: (a) Mutation (b) Gene amplification (c) Chromosome rearrangements. These mechanisms result in either an increase in protooncogene expression or a change in protooncogene structure [47]. The EBV-mediated B-cell change is associated with a global improvement in viral and cellular expression of genes. The biologic characteristics of the virus were instantly fascinating, as it was shown that cell lines could be determined from samples of Burkitt's lymphoma (BL) and could propagate a virus that could strike primary B cells with EBV and turn them into immortalized cell lines [48]. This study of molecular phenotype led to the discovery of viral proteins that are necessary for latent infection and needed for cell transformation [49]. The mechanisms of EBV oncogenesis include:

#### *1.2.7.1 Alteration of host cellular signaling pathways*

EBV encoding oncogenes induce the changes in the host cellular signaling pathways that control proliferation, differentiation, cell death, genomic integrity, and recognition by the immune system.

LMP1, LMP2A, and LMP2B, latent membrane proteins are generated of the common viral locus with converging and interfering primary transcripts [50]. The LMP1 is one of the main EBV-encoded oncoproteins and it is a constitutively active mimic of cellular CD40 receptor. It is critically important for the EBV-induced B-cell transformation via the activation of NF-κB, c-Jun N-terminal kinase (JNK), and p38 cascades [21]. LMP1 also regulates cellular apoptosis by triggering the NF-κB pathway by increasing the antiapoptotic expression of Bcl2 via IRAK1 and TRAF6 whereIRAK1 is necessary for both p38 and p65/RelA phosphorylation [50]. Also, LMP1-stimulated proapoptotic polycomb complex protein (Bmi-1) is further been recruiting by EBNA3C for the transcriptional funnel of other genes. LMP2A acts as a functional homolog of the B-cell receptor (BCR) and thus promotes the survival of B-cells. Likewise, it is essential for the growth transformation of germinal center-derived B-cells which are BCR negative [21]. LMP2B negatively regulates LMP2A functions and transition from latent to lytic activation by depleting LMP2A-mediated BCR cross-linking and restoring Ca2+ mobilization [51].

EBNA1 is important for the DNA replication and maintenance of the viral latent genome. It binds to the viral episomal replication origin (OriP) and simultaneously to the host cell hromosomes that enable viral genome duplication during each cell cycle [22]. Through promotor selection, combined with comprehensive epigenetic control, EBNA1 can organize the shift between different latency programs, and EBNA1 can produce transcripts for different cells and help improve the control of telomeres on cell chromosomes [52]. The p53 and Mdm2 affected by the EBNA1 binding with ubiquitin-specified protease 7 (USP7), contributes to antiapoptotic activity control, likely by promoting survivin expression levels [23, 53].

EBNA2 and EBNALPare the first latent genes expressed following B-cell infection. EBNA2 is the main viral transcription factor responsible for activation of the expression of the entire repertoire of latent transcripts along with several host genes, utilizing cell transcription factors, RBP-J and EBF1 [22]. At the same time, EBNALP supports transcriptional regulation by EBNA2 via blocking off the activity of NCoR and RBP-J [52]. The EBNA2 contributes most strongly to the proliferation of B-cells through the activation of about 300 cell genes, such as the transcription of MYC and RUNX3 [52].

EBNA3 protein family consisting of EBNA3A, -3B, and -3C are transcription factors that precisely regulate host gene transcription and the proliferation of B-cells, particularly in the immunosuppression environment. Also, EBNA3B knockout virus-induced tumors demonstrated a lack of T-cell infiltrate and related CXCL10 chemokine activation [53]. In comparison, EBNA3A and EBNA3C cooperate as predominant viral oncoproteins by controlling the transcription of the cellular gene. This phenomenon is also true for EBNA3A [54].

The EBNA3A and EBNA3C have been demonstrated to react with a long list of cellular proteins and transcription factors involved in the regulation of multiple cell signaling pathways [55]. Interactive partners for EBNA3C involve transcription factors, chromatin modulators (both histone deacetylase and histone acetylase enzymes), cell-cycle proteins including G1-S and G2-M transitions, metastases suppressors, post-translational modifiers, E3-ubiquitin ligase, ubiquitin-specific proteases, unfolded protein response (UPR) regulators, cell tumor suppressors, and oncoproteins [56]. The EBNA3C has been shown to form a complex with Chk2 and thus manipulates the G2/M step of the cell cycle [54]. Overall, the B-cell transformation and B-cell lymphoma are directly affected by the EBNA3 protectors by targeting main cell signaling cascades including cell cycle, apoptosis, and autophagy [56].

• Noncoding viral transcripts.

A variety of noncoding RNAs (ncRNAs) in EBV infected B cells can be expressed, known as the EBV-encoded non-polyadenylated RNAs (EBER1 and EBER2) and numerous miRNAs [57]. Such ncRNAs are not necessary for the transformation of B-cells, but they are associated with immune evasion, and demonstrated in various forms of latency systems. In addition, EBER in situ hybridization is the most reliable and sensitive method to detect EBV infection in tissues of various EBV-related malignancies. EBER expression promotes the growth of B cells by blocking of PKR phosphorylation and inhibition of translational initiation factor eIF-2a and alpha-interferon (IFN-α)-induced apoptosis [56]. EBER can interact with ribosomal protein L22 that regulates protein translation, gene expression and PKR dependent apoptosis [58]. The EBER2 directly recruits PAX5 for the control of LMP2A expression, which has been verified by the usage of the EBER2 mutant virus with lower LMP2A expression [58]. EBV encodes more than 40 mature miRNAs, which are encoded at 2 different loci in the EBV genome:

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**Figure 5.**

*lymphoma.*

*Human Herpetic Viruses and Immune Profiles DOI: http://dx.doi.org/10.5772/intechopen.96340*

an epithelial cell [59].

BHRF1 locus (BHRF1 miRNAs) and BART locus (BART miRNAs). The expression of various EBV miRNA is different among different cells. Viral miRNAs can either target other EBV transcripts or transcripts of host cells. BHRF1 miRNAs exhibit expression that is restricted to latency 3 whereas the BART miRNAs are expressed in all latency types. The expression of BHRF1 miRNAs in infected B lymphocytes, target multiple tumor suppressor proteins such as PTEN and P27KIP1 for the B-cell transformation. Viral miRNAs also inhibit the expression of several tumor suppressor genes, including DICE1, PUMA, PTEN and BCL2L11 to promote the survival of

**Table 2** and **Figure 5** explain the key latent transcription mechanisms of EBV.

*Special features of EBV latent transcripts during B-cell transformation associated in the development of B-cell* 

#### *Human Herpetic Viruses and Immune Profiles DOI: http://dx.doi.org/10.5772/intechopen.96340*

BHRF1 locus (BHRF1 miRNAs) and BART locus (BART miRNAs). The expression of various EBV miRNA is different among different cells. Viral miRNAs can either target other EBV transcripts or transcripts of host cells. BHRF1 miRNAs exhibit expression that is restricted to latency 3 whereas the BART miRNAs are expressed in all latency types. The expression of BHRF1 miRNAs in infected B lymphocytes, target multiple tumor suppressor proteins such as PTEN and P27KIP1 for the B-cell transformation. Viral miRNAs also inhibit the expression of several tumor suppressor genes, including DICE1, PUMA, PTEN and BCL2L11 to promote the survival of an epithelial cell [59].

**Table 2** and **Figure 5** explain the key latent transcription mechanisms of EBV.

#### **Figure 5.**

*Special features of EBV latent transcripts during B-cell transformation associated in the development of B-cell lymphoma.*

#### *1.2.7.2 Suppression, escape, and modulation of host immunity*

Multiple genes have been reported to suppress antigen presentation. EBNA1 contains a Gly/Ala repeat sequence, through which proteasomal degradation and antigen presentation of the protein is impaired, while BNLF2A targets the transporter associated with antigen processing and blocks antigen presentation. BGLF5 represses HLA class I synthesis, whereas BILF1 downregulates cell surface expression of the molecule. It is highly likely that at least LMP1 and LMP2A, the viral functional mimic of CD40 and BCR, have tactfully evolved to modify those processes in the germinal center, and thus, these EBV gene products can deregulate the immune system for survival [60]. See section 1.1.8.

#### *1.2.7.3 Genetic or epigenetic background/alteration of the host genome*

Virally-induced epigenetic alterations of the host genome are evident in EBVassociated cancers, which are the result of genetic mutations, changes involving DNA methylation and chromatin structure that in turn alter the expression of growth promoting or suppressing genes. Enhanced Ig-Myc translocation has been demonstrated in Burkitt lymphoma [34, 35] and silencing of tumor suppressor genes (e.g. p16INK4A) in many EBV-positive cancers. Several reports have demonstrated that the predisposition of individual HLA allele significantly affects the morbidity of EBV-positive proliferative disorders, particularly in NPC and Hodgkin lymphoma [37–42]. LMP1 seems to induce genetic/epigenetic alterations by DNA hypermethylation and chromatin modifications [51].

#### *1.2.8 Immune evasion mechanisms by Epstein–Barr virus*

In response to primary EBV infection, both innate and adaptive antiviral responses have been activated. Despite the very effective immune response, the virus is not cleared.. A lifelong, latent infection is established within the memory B-cell and EBV genomes are propagated during the division of the transformed, latently infected B cells. During this period, broad range of EBV early gene products interfere with immune response which helps the virus to persist and to reactivate [60].

#### *1.2.8.1 Evasion of innate immunity*

Various pattern recognition receptors (PRRs) including cell surfaces and endosomal Toll- receptors (TLRs) and cytoplasmic DNA and RNA sensors are capable of detecting EBV particles. The EBV is identified by Toll-like receptors (TLRs) on the cell surface and in endosomes [61]. Virus-derived or virus-induced components can be detected by RNA and DNA sensors as well as by inflammasomes within the cytosol. TLRs, RNA and DNA sensors trigger a cascade of intracellular signaling events that enable the activation of the interferon regulatory factors (IRFs) and NF-κB. As a result, activated gene transcriptions induce the production of cytokines and type I interferon (IFN I). Different levels of these PRR signals are attacked by latent and/ or lytic EBV proteins or EBV miRNA as recently seen [62, 63].

#### *1.2.8.1.1 Reduction of Toll-like receptor expression*

EBV can inhibit the synthesis of cellular proteins in infected cells through global mRNA destabilization. This mechanism is via the EBV DNase (alkaline

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and RNA substrates [62].

decreasing IFNβ expression [64].

the minimal amount of the NF-κB [65].

*1.2.8.2 Evasion of adaptive immunity*

*1.2.8.2.1 Evasion of CD8+ T Lymphocytes*

*1.2.8.1.4 Interference with innate effector molecules*

exonuclease) BGLF5 regulated by the supplementary BGLF 5 RNase feature, which is expressed during the active period of infection and uses the same catalytic position as DNase action, but the substrate-bindings site is only partly shared by DNA

A significant number of lytic EBV proteins interact with host IRFs, which are the transcription factors that stimulate the synthesis of type IFN. The immediate-early EBV transactivator BZLF1, BRLF1 and tegument protein LF2 interact with IRF7 andIRF3, and inhibit its transcriptional activity on IFNα4 and IFNβ promoters resulting in the suppression of antiviral state induction. In addition, EBV protein kinase BGLF4 phosphorylates and inhibits IRF3 transcriptional activity, thus

The EBV infection is linked to the decreased NF-κB--dependent gene expression. The expression of viral BZLF1 and cellular NF-κB is reciprocally inhibited. The higher levels of the NF-κB in the absence of BZLF1 instigate EBV latency, while increased expression of the BZLF1 after the induction of the lytic cycle overwhelms

Several EBV gene products have an impact on the function of effector molecules of innate immunity. The host cytokine colony-stimulating factor 1 (CSF-1) activates the differentiation of the macrophage, and the secretion of the IFN-α, EBV encodes the soluble form of the CSF-1 receptor BARF1, which neutralizes the effects of the host CSF, leading to the reduction of the IFN-secretion of EBV infected mononuclear cells. EBV BZLF1 counteracts intrinsic effector molecules in a variety of ways [62]. First, BZLF1 decreases TNF5–007 and IFNΔ receptors to minimize cellular susceptibility to these cytokines; second, BZLF1 induces SOCS3-signaling cytokine suppressor, which inhibits JAK/STAT signaling and thus promotes IFNresponsiveness Type I state; Third, BZLF1 triggers TGFβ immunosuppressive cytokine expression and disrupts the development of Promyelocytic leukemia

The EBV compromises the activation of both CD8 + and CD4 + T cells by interfering in different stages of HLA Class I and Class II antigen presentation pathways,

The EBV encodes at least three proteins that independently interfere with antigen presentation through deregulation of the surface expression of HLA I in many ways to prevent EBV-specific (memory) T cell recognition [68]. BGLF5 induces degradation of HLA I-encoding mRNA and reduces the presence of peptide at the cell surface which inhibits T-cell recognition. Ut has been suggested that BNLF2adeplete peptides from the ER (HLA I loading compartment) and inhibits the importation of peptides by the antigen- transporter (TAP). BILF1, encoding a

*1.2.8.1.2 Modulation of IRF signaling and Type I interferon production*

*1.2.8.1.3 Interference with NF-*κ*B and inflammatory pathways*

bodies (PML-bodies) that may have antiviral activity [66].

especially during the productive phase of infection [67].

#### *Human Herpetic Viruses and Immune Profiles DOI: http://dx.doi.org/10.5772/intechopen.96340*

exonuclease) BGLF5 regulated by the supplementary BGLF 5 RNase feature, which is expressed during the active period of infection and uses the same catalytic position as DNase action, but the substrate-bindings site is only partly shared by DNA and RNA substrates [62].

#### *1.2.8.1.2 Modulation of IRF signaling and Type I interferon production*

A significant number of lytic EBV proteins interact with host IRFs, which are the transcription factors that stimulate the synthesis of type IFN. The immediate-early EBV transactivator BZLF1, BRLF1 and tegument protein LF2 interact with IRF7 andIRF3, and inhibit its transcriptional activity on IFNα4 and IFNβ promoters resulting in the suppression of antiviral state induction. In addition, EBV protein kinase BGLF4 phosphorylates and inhibits IRF3 transcriptional activity, thus decreasing IFNβ expression [64].

#### *1.2.8.1.3 Interference with NF-*κ*B and inflammatory pathways*

The EBV infection is linked to the decreased NF-κB--dependent gene expression. The expression of viral BZLF1 and cellular NF-κB is reciprocally inhibited. The higher levels of the NF-κB in the absence of BZLF1 instigate EBV latency, while increased expression of the BZLF1 after the induction of the lytic cycle overwhelms the minimal amount of the NF-κB [65].

#### *1.2.8.1.4 Interference with innate effector molecules*

Several EBV gene products have an impact on the function of effector molecules of innate immunity. The host cytokine colony-stimulating factor 1 (CSF-1) activates the differentiation of the macrophage, and the secretion of the IFN-α, EBV encodes the soluble form of the CSF-1 receptor BARF1, which neutralizes the effects of the host CSF, leading to the reduction of the IFN-secretion of EBV infected mononuclear cells. EBV BZLF1 counteracts intrinsic effector molecules in a variety of ways [62]. First, BZLF1 decreases TNF5–007 and IFNΔ receptors to minimize cellular susceptibility to these cytokines; second, BZLF1 induces SOCS3-signaling cytokine suppressor, which inhibits JAK/STAT signaling and thus promotes IFNresponsiveness Type I state; Third, BZLF1 triggers TGFβ immunosuppressive cytokine expression and disrupts the development of Promyelocytic leukemia bodies (PML-bodies) that may have antiviral activity [66].

#### *1.2.8.2 Evasion of adaptive immunity*

The EBV compromises the activation of both CD8 + and CD4 + T cells by interfering in different stages of HLA Class I and Class II antigen presentation pathways, especially during the productive phase of infection [67].

#### *1.2.8.2.1 Evasion of CD8+ T Lymphocytes*

The EBV encodes at least three proteins that independently interfere with antigen presentation through deregulation of the surface expression of HLA I in many ways to prevent EBV-specific (memory) T cell recognition [68]. BGLF5 induces degradation of HLA I-encoding mRNA and reduces the presence of peptide at the cell surface which inhibits T-cell recognition. Ut has been suggested that BNLF2adeplete peptides from the ER (HLA I loading compartment) and inhibits the importation of peptides by the antigen- transporter (TAP). BILF1, encoding a

constitutively active G protein-coupled receptors (GPCR) which reduce the transportation of HLA I from the trans-Golgi network. In addition, cell surface turnover is increased and subsequently i degradation by lysosomal proteases. These proteins are expressed during the replicative process of EBV and function in tandem with the prevention of CD8 + T cells being recognized [67].

#### *1.2.8.2.2 Evasion of CD4+ T Lymphocytes*

The EBV has adopted several strategies for immune evasion that interfere with CD4 + T-cell immunity. The EBV receptor Gp42 can bind to the B-cell HLA class II molecules. The HLA Class II/peptide complex relationship blocks T-cell receptor (TCR)—class II interactions and prevents CD4 + T cell activation. Besides, protein GP42/gH/gLdecreases the HLA II cell surface expression by the HLA II mRNA degradation. Inhibiting the activities of CIITA promoters and, as a result, lowering the HLA II surface levels, EBV also encodes a viral IL-10 homolog (BCRF1) that has been identified as impairing the IFNβ signal. The IL-10 is an anti-inflammatory cytokine that can inhibit CD4 + priming and effector functions and modulates them; BCRF1 was suggested to inhibit CD4 + T-cell antiviral response similar to IL-10 [69].

#### *1.2.8.2.3 Immune evasion during latency*

The EBV severely restricts latent infection viral protein expression to prevent host immune recognition. Different latency forms represent different stages from primary B cell infection to the transformation of the growth. Thus, in latency III cells, EBNA1inhibits its translation and proteasomal degradation. This strategy ensures adequate levels of EBNA1 to preserve the viral genome while decreasing the turnover of proteins to minimize the appearance of viral antigens to CD8 + T cells. LMP1 and 2 mediate NF-κB activation and decrease the TLR9 surface expression and accelerate the turnover of IFN receptors, resulting in a decrease in the incidence of IFN receptors. During latency II, the expression is limited to EBNA1 and LMP1, and 2. Latency I only contains an expression of EBNA1, and latency 0 occurs without any expression of EBV protein [70]. The EBV encodes different types of non-coding RNAs, including two EBV-encoded small RNAs (EBERs) that inhibit PKR activity and miRNAs that de-regulate T-cell attracting CXCL-11 chemokines and de-regulates T-bet and IFNΔ transcriptional regulator [71].

#### *1.2.9 EBV prevention and vaccine*

In the vast majority of individuals, EBV is a harmless passenger, controlled easily by immune defenses, but in some individuals, EBV drives a broad range of diseases that can cause significant morbidity and mortality.

A vaccine is currently unavailable. A prophylactic vaccine which prevents acute disease, the most beneficial using the humoral immune approach, vaccines expressing the major viral envelop protein, gp350 have been developed. Most recently live recombinant vaccinia vaccine expressing gp350 protected two- thirds of the vaccinated infants [20]. as Also, therapeutic vaccines are investigated. These vaccines are based on direct peptide immunization approaches. The use of immunodominant HLA Class I and II epitopes of LMP1, LMP2, and EBNA1 may induce a strong and sustained T-cell response, which was demonstrated with some success primarily in reactivating CD4+ and CD8+ cell in vitro [72, 73].

The use of antiviral therapy in EBV infection is limited. Antiviral therapy can be used as preemptive therapy of PTLD in EBV- organ transplant recipients. These

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**1.3 Human Cytomegalovirus (HCMV)**

*1.3.1 The virion and genome organization*

infected B-lymphocytes [32].

agents can block EBV replication in donor B-cells and infection of recipient B-cells. Prophylactic intravenous ganciclovir after liver transplantation lead to decreasing of PTLD incidence, which may be due to a reduction in the number of latently-

Human Cytomegalovirus (HCMV) or human herpesvirus 5 (HHV-5) [74], is ubiquitous in human populations and was first isolated and cultivated in 1956 [56]. The HCMV derives its name from the Greek cyto-, "cell", and -megalo-, "large", because of the enlargement of virus infected cells, (cytomegaly), [75]. Human cytomegalovirus is a leading - cause of congenital viral infections with numerous consequences such as birth defects including intrauterine growth restriction, stillbirth, low birth weight, preterm birth, microcephaly, neurodevelopmental delay, cerebral palsy, hematological disorders, pneumonitis, blindness, and sensorineural hearing loss [76]. HCMV infection is typically clinically silent in immunocompetent hosts, with few mild symptoms like fever, myalgia andcervical lymphadenopathy. Individuals with weakened immunity – neonates, diagnosed with HIV/AIDS, and those on long-term immunosuppressive treatments, such as transplant recipients – HCMV infection often results in clinically severe diseases. The worst outcomes, including mortality and long-term morbidity, are with congenital infection [56].

A complete virus particle consists of a DNA core with a protein coat and external envelopes representing the extracellular infective form of a virus called virion [77]. The average size of viral particle varies between 200–300 nanometers and has linear double-stranded (235-kb) DNA genome which is enclosed by an icosahedral capsid. The capsid's diameter is (110–130 nm) and made of 162 capsomeres (12 pentons and 150 hexons).. HCMV has three kinds of capsids: A capsid (only capsid shell), B capsid (capsid shell and assembled proteins), and C capsid (a mature capsid containing the viral genome). These three capsids represented in three various stages of capsid maturation that takes place in the nucleus of infected cells capsid is composed of at least 5 proteins, i- Major Capsid Protein (UL86), ii- Minor Capsid Protein (UL85), iii- Smallest Capsid Protein (SCP, UL48–49), iv- Assembly protein (Fragments of

The widest layer inside the virion structure is the tegument layer that closely surrounds the capsid; anchoring the envelope to the tégumented capsid is believed to be essential. The layer of tegument is composed of several proteins like pp65/ ppUL83, pp71/ppUL82, pp150/pUL32, and pp28/pUL99 which play main roles during the entry of virus (un-coating), intracellular capsid transportation and assembly [79]. The tegument is covered by a lipid bilayer called the envelope that keeps the entire virion intact. It interacts with the host cell membrane in target cells and thus plays a significant role in the attachment and entry of viruses. Lipid bilayer envelope is derived from cellular ER or endosomal membranes and associated with 23 viral glycoproteins. The viral glycoproteins gpUL55 (gB), gpUL73 (gN), gpUL74 (gO), gpUL75 (gH), UL100 (gM), gpUL115 (gL) and the pentameric complex consist of gL, gH and UL128–131, are known to play crucial roles in viral entry, cell-

Among herpesviruses, HCMV has the largest genome consisting of a linear dsDNA ranging from 220 to 240 kbp and comprising up to 250 opening reading frames (ORFs) [81]. The herpesvirus genomes are categorized into A-F sections depending on the organization of the genome segments (**Figure 7**). The genome of

UL80) and v- Minor Capsid Binding Protein (MCP, UL46) [78].

to-cell spread and virion maturation [80] (**Figure 6**).

agents can block EBV replication in donor B-cells and infection of recipient B-cells. Prophylactic intravenous ganciclovir after liver transplantation lead to decreasing of PTLD incidence, which may be due to a reduction in the number of latentlyinfected B-lymphocytes [32].

#### **1.3 Human Cytomegalovirus (HCMV)**

Human Cytomegalovirus (HCMV) or human herpesvirus 5 (HHV-5) [74], is ubiquitous in human populations and was first isolated and cultivated in 1956 [56]. The HCMV derives its name from the Greek cyto-, "cell", and -megalo-, "large", because of the enlargement of virus infected cells, (cytomegaly), [75]. Human cytomegalovirus is a leading - cause of congenital viral infections with numerous consequences such as birth defects including intrauterine growth restriction, stillbirth, low birth weight, preterm birth, microcephaly, neurodevelopmental delay, cerebral palsy, hematological disorders, pneumonitis, blindness, and sensorineural hearing loss [76]. HCMV infection is typically clinically silent in immunocompetent hosts, with few mild symptoms like fever, myalgia andcervical lymphadenopathy. Individuals with weakened immunity – neonates, diagnosed with HIV/AIDS, and those on long-term immunosuppressive treatments, such as transplant recipients – HCMV infection often results in clinically severe diseases. The worst outcomes, including mortality and long-term morbidity, are with congenital infection [56].

#### *1.3.1 The virion and genome organization*

A complete virus particle consists of a DNA core with a protein coat and external envelopes representing the extracellular infective form of a virus called virion [77]. The average size of viral particle varies between 200–300 nanometers and has linear double-stranded (235-kb) DNA genome which is enclosed by an icosahedral capsid. The capsid's diameter is (110–130 nm) and made of 162 capsomeres (12 pentons and 150 hexons).. HCMV has three kinds of capsids: A capsid (only capsid shell), B capsid (capsid shell and assembled proteins), and C capsid (a mature capsid containing the viral genome). These three capsids represented in three various stages of capsid maturation that takes place in the nucleus of infected cells capsid is composed of at least 5 proteins, i- Major Capsid Protein (UL86), ii- Minor Capsid Protein (UL85), iii- Smallest Capsid Protein (SCP, UL48–49), iv- Assembly protein (Fragments of UL80) and v- Minor Capsid Binding Protein (MCP, UL46) [78].

The widest layer inside the virion structure is the tegument layer that closely surrounds the capsid; anchoring the envelope to the tégumented capsid is believed to be essential. The layer of tegument is composed of several proteins like pp65/ ppUL83, pp71/ppUL82, pp150/pUL32, and pp28/pUL99 which play main roles during the entry of virus (un-coating), intracellular capsid transportation and assembly [79]. The tegument is covered by a lipid bilayer called the envelope that keeps the entire virion intact. It interacts with the host cell membrane in target cells and thus plays a significant role in the attachment and entry of viruses. Lipid bilayer envelope is derived from cellular ER or endosomal membranes and associated with 23 viral glycoproteins. The viral glycoproteins gpUL55 (gB), gpUL73 (gN), gpUL74 (gO), gpUL75 (gH), UL100 (gM), gpUL115 (gL) and the pentameric complex consist of gL, gH and UL128–131, are known to play crucial roles in viral entry, cellto-cell spread and virion maturation [80] (**Figure 6**).

Among herpesviruses, HCMV has the largest genome consisting of a linear dsDNA ranging from 220 to 240 kbp and comprising up to 250 opening reading frames (ORFs) [81]. The herpesvirus genomes are categorized into A-F sections depending on the organization of the genome segments (**Figure 7**). The genome of

**Figure 6.** *Structure of an HCMV Virion.*

**Figure 7.** *The six classes of herpesvirus genomes.*

HCMV is classified as an E genome. The sequencing analysis of the HCMV genome has shown that it has a very complex structure. Generally, the genome is organized into two parts: The single long regions (UL) and unique small regions (US) flanked by terminal repeats (TR) and internal repeats (IR), UL-area ORFs and US-region ORFs are classified according to their location [82] (**Figure 8**). More than 70 viral proteins have been identified from the purified virions [83]. Only 50 proteins are important for viral replication, while the vast majority of HCMV proteins are involved in host immunomodulation via their interference with cellular signals [84]. HCMV encodes for at least 4 long polyad-encoded RNAs and 26 microRNAs which have major functions during host-virus and virus replication interactions [85].

#### *1.3.2 Life cycle of human Cytomegalovirus*

#### *1.3.2.1 Entry*

Cytomegalovirus virus (CMV) enters host cells through membrane fusion as shown in **Figure 9**. The viral entry involves the binding of viral glycoproteins on the surface of the viral lipid envelope and the specific receptors on the external membrane of the host cell [86]. This initial interaction makes the cell susceptible to further interactions that fuse the membranes and eventually disassemble and release the viral genomic DNA into the host cell. Many tegument proteins are thought to mediate the delivery of the DNA-containing nucleocapsid to the nuclear pore complex and the release of the viral DNA into the nucleus [87].

HCMV-gB mediates attachment to cells via binding to cellular receptors that include heparan sulfate proteoglycan, integrins, and epidermal growth factor receptor (EGFR) to promote the entrance process [9]. The heparan sulfate proteoglycan

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**Figure 9.**

*Overview of the human cytomegalovirus life cycle.*

**Figure 8.**

molecule permits the HCMV particle to attach closer to the cell membrane where viral glycoproteins bind to more specific receptors. This is mediated by gB and gH or their complexes, and many other viral protein complexes may also mediate the process of entry; a homodimer of gB known as gC-I, and a heterodimer composed of gM and gN which form gC-II and a heterotrimer of gH, gL and gO which form the gC-III complex. HCMV has two different routes of entry in different cell types [88]. Virus entry in fibroblasts is mediated by viral envelope glycoprotein complexes gH/ gL-gB and gH/gL/gO via direct fusion at the plasma membrane. Aminopeptidase N (CD13) and Annexin-II may also serve as receptors to promote entry at the plasma membrane. The entry of CMV into other relevant cell types, such as endothelial

*Human Herpetic Viruses and Immune Profiles DOI: http://dx.doi.org/10.5772/intechopen.96340*

*Illustration of the CMV life cycle from viral entry to egress of new infectious virions.*

#### *Human Herpetic Viruses and Immune Profiles DOI: http://dx.doi.org/10.5772/intechopen.96340*

#### **Figure 8.**

*Illustration of the CMV life cycle from viral entry to egress of new infectious virions.*

molecule permits the HCMV particle to attach closer to the cell membrane where viral glycoproteins bind to more specific receptors. This is mediated by gB and gH or their complexes, and many other viral protein complexes may also mediate the process of entry; a homodimer of gB known as gC-I, and a heterodimer composed of gM and gN which form gC-II and a heterotrimer of gH, gL and gO which form the gC-III complex. HCMV has two different routes of entry in different cell types [88]. Virus entry in fibroblasts is mediated by viral envelope glycoprotein complexes gH/ gL-gB and gH/gL/gO via direct fusion at the plasma membrane. Aminopeptidase N (CD13) and Annexin-II may also serve as receptors to promote entry at the plasma membrane. The entry of CMV into other relevant cell types, such as endothelial

cells, follows an endocytic route, clathrin is the major constituent of coated vesicles and plays an important role in the endocytic entry of viruses [89]. HCMV has a wide cell tropism and can infect different cell types [34], such as neuronal cells in the brain and retina [90], fibroblasts, epithelial and endothelial cells (EC) in lung and gastrointestinal tract [91], hepatocytes in the liver [90] and peripheral blood mononuclear lymphocytes (PBML) [92]. Infected monocytes can release infectious viruses into target organs through tissue-macrophage differentiation [93]. Efficient infection of EC of blood vessel may lead to separation of these cells and hematogenic dissemination may, therefore, be initiated [94]. Fibroblasts, on the other hand, are likely to contribute to the efficient development of HCMV and will help the establishment of the primary infection. HCMV can also infect various kinds of cells in vitro. However, the replication of viruses varies between different cell types. In fibroblasts, smooth muscle cells, endothelial cells, and epithelial cells, HCMV inducesproductive infection, while in poorly differentiated cells, such as myeloid-linear progenitor cells, viral replication is limited [91]. Fibroblasts are the most commonly used cells for HCMV cultivation in the laboratory because HCMV binds to fibroblasts with the efficiency of 2500–3000 particles per cell [94].

HCMV strains show broad variations in the relative pentamer and trimer rates of viral glycoproteins incorporated into virions, which correspond to the cell tropism differences between strains [93]. Many HCMV genes are capable of influencing viral cell tropism at the entry stage and most likely function by composite effects of gH/gL complexes.

#### *1.3.2.2 Lytic replication*

HCMV infection of the cell leads to an active replication with the production of new viral particles that are released by exocytosis of the infected cells, known as the lytic phase [95]. The replication of viral DNA is dependent on the expression of certain viral proteins [96]. Gene expression of viral DNA takes place in three stages: immediate early genes (IE) expression, followed by early genes (E) and late genes (L) expressions [95]. The IE gene products act as transcription factors as well as trans-activators to regulate expression of the E and L genes. Two predominant nuclear phosphoproteins, IE1-p72 (Immediate Early 1 – Protein 72KD) and IE2-p86 (Immediate Early 2- Protein 86KD) have the key roles., IE1-p72 is expressed from the UL123 ORF during the IE phase of replication cycle. IE1-p72 transactivates the promoters of numerous HCMV early genes including gene products that facilitate the replication process. It also interacts with the p107 protein through a domain at the N-terminus of IE1-p72 and increase the p107- mediated repression of E2F promoters leading to the inhibition of p107- mediated growth suppression [97]. Therefore, it appears that IE1-p72 can induce E2F activity. The expression of IE1 p72 can promote S phase entry only in cells lacking p53 or p21 [96]. Controversial to that, IE1- p72 expression causes wild-type cells to arrest, most likely in G1 due to increased levels of p53 protein, which results in a p53-dependent induction of p21 expression and subsequent growth arrest [96, 97]. IE2-p86 is expressed from the UL122 ORF during the IE phase of the replication cycle and is essential for HCMV replication [98]. The protein IE2-p86 specifically interacts with pRb through more than one domain and induce pRb mediated repression of E2F promoters. The IE2-p86 disruption of pRb-E2F complexes enable E2Fs transactivation of its target genes [99]. However, IE2-p86 induces cells to enter S phase, an effect that could be attributed in part to IE2-p86 transactivation of the cyclin E promoter and induction of E2F activity [100].

Phosphoprotein 65 (pp65) is a tegument phosphoprotein that exhibits kinase activity. This protein may affect the activity of a specific subset of cytotoxic

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into lytic replication and new viral progeny occurs [101].

*1.3.2.3 Virus assembly*

*1.3.2.4 Latency and reactivation*

T –Lymphocytes (CTLs) by modification of IE1- p72, possibly through its phosphorylation and may interfere with its processing and/or degradation [82]. Following peak expression of IE regulatory proteins, early genes (E genes) become transcriptionally active (**Figure 9**). These proteins regulate replication process of HCMV DNA, such as a DNA-polymerase (UL54) and DNA primase (UL70) which sustain an efficient production of new viral progeny [89]. The L proteins, which are mainly structural components, are essential for virion assembly and egress. HCMV genome contains a cis-acting lytic origin of DNA replication (OriLyt) element to initiate bidirectional DNA replication (theta form of replication), followed by a rolling circle mode of replicationof viral DNA molecules and their incorporation into new virus particles [80]. The entire replication cycle for HCMV takes approximately 72 h and the mature new virions infect the new cells either by their release from the infected cells or by the spread via cell-to-cell mechanisms [80]. During latency, only selected IE gene transcription and translation of viral proteins occur and when conditions are favorable, the virus may be reactivated

The newly synthetized DNA is inserted into an immature B capsid after the precapsid assembly stage and becomes a fully mature C capsid. This DNA packed capsid, egresses through the nuclear membrane from the nucleus, through an envelope and de-envelopment cycle [102]. The mechanism of assembly of tegument proteins is still unclear. It has been suggested that tegument proteins are added to nucleocapsids sequentially starting in the nucleus and continuing in the cytoplasm, which provide stability during nucleocapsid translocation from the nucleus to the cytoplasm.. The final envelopment of tegumented particle occurs at ER/endosomal membranes. By transporting Rab3 secretory vesicles, mature particles are released by fusion of the vacuole with the plasma membrane and shed out by exocytosis [103] (**Figure 9**).

The establishment of latency is one of the major biological characteristics of herpesviruses. Primary HCMV infection is often asymptomatic in a healthy person (immune-competent host) and leads to latent and recurrent infection [104].

The MIE gene acts as a transactivator for transcribing the majority of encoded HCMV genes and is necessary for the virus replication and the lytic process of infection. During latency, cellular factors transcripts control the MIE promoter. These factors also suppress the chromatin around the MIE gene, which prevents the lytic cycle stage and is a part of preserving the latent stage of the infection [105]. People who have had an organ or bone marrow transplant and those with AIDS can develop serious illness caused by CMV. Typically, latent virus from a previous infection (the primary CMV infection may have occurred many years earlier) becomes active again because the person's immune system is compromised [106]. To establish viral latency and maintenance, the latency- gene product UL138 is required. HCMV-LUNA and UL138 are generated during HCMV latency and lytic infection and activate CD4 + T cells, resulting in the development of IL-10 and IFN-π; one with immunosuppressive effects and one with immune-activating effects that facilitate the replication and reactivation of latent HCMV. The produced IFN-β can promote macrophage differentiation, which can lead to reactivation and enhanced HCMV replication. UL138 also downgrades protein-1, MRP, which can lead to lower exports of C4 cell leukotriene, preventing DCs from entering lymph

nodes and impairing an HCMV-specific immune response [105].

#### *Human Herpetic Viruses and Immune Profiles DOI: http://dx.doi.org/10.5772/intechopen.96340*

T –Lymphocytes (CTLs) by modification of IE1- p72, possibly through its phosphorylation and may interfere with its processing and/or degradation [82].

Following peak expression of IE regulatory proteins, early genes (E genes) become transcriptionally active (**Figure 9**). These proteins regulate replication process of HCMV DNA, such as a DNA-polymerase (UL54) and DNA primase (UL70) which sustain an efficient production of new viral progeny [89]. The L proteins, which are mainly structural components, are essential for virion assembly and egress. HCMV genome contains a cis-acting lytic origin of DNA replication (OriLyt) element to initiate bidirectional DNA replication (theta form of replication), followed by a rolling circle mode of replicationof viral DNA molecules and their incorporation into new virus particles [80]. The entire replication cycle for HCMV takes approximately 72 h and the mature new virions infect the new cells either by their release from the infected cells or by the spread via cell-to-cell mechanisms [80]. During latency, only selected IE gene transcription and translation of viral proteins occur and when conditions are favorable, the virus may be reactivated into lytic replication and new viral progeny occurs [101].

#### *1.3.2.3 Virus assembly*

The newly synthetized DNA is inserted into an immature B capsid after the precapsid assembly stage and becomes a fully mature C capsid. This DNA packed capsid, egresses through the nuclear membrane from the nucleus, through an envelope and de-envelopment cycle [102]. The mechanism of assembly of tegument proteins is still unclear. It has been suggested that tegument proteins are added to nucleocapsids sequentially starting in the nucleus and continuing in the cytoplasm, which provide stability during nucleocapsid translocation from the nucleus to the cytoplasm.. The final envelopment of tegumented particle occurs at ER/endosomal membranes. By transporting Rab3 secretory vesicles, mature particles are released by fusion of the vacuole with the plasma membrane and shed out by exocytosis [103] (**Figure 9**).

#### *1.3.2.4 Latency and reactivation*

The establishment of latency is one of the major biological characteristics of herpesviruses. Primary HCMV infection is often asymptomatic in a healthy person (immune-competent host) and leads to latent and recurrent infection [104].

The MIE gene acts as a transactivator for transcribing the majority of encoded HCMV genes and is necessary for the virus replication and the lytic process of infection. During latency, cellular factors transcripts control the MIE promoter. These factors also suppress the chromatin around the MIE gene, which prevents the lytic cycle stage and is a part of preserving the latent stage of the infection [105].

People who have had an organ or bone marrow transplant and those with AIDS can develop serious illness caused by CMV. Typically, latent virus from a previous infection (the primary CMV infection may have occurred many years earlier) becomes active again because the person's immune system is compromised [106].

To establish viral latency and maintenance, the latency- gene product UL138 is required. HCMV-LUNA and UL138 are generated during HCMV latency and lytic infection and activate CD4 + T cells, resulting in the development of IL-10 and IFN-π; one with immunosuppressive effects and one with immune-activating effects that facilitate the replication and reactivation of latent HCMV. The produced IFN-β can promote macrophage differentiation, which can lead to reactivation and enhanced HCMV replication. UL138 also downgrades protein-1, MRP, which can lead to lower exports of C4 cell leukotriene, preventing DCs from entering lymph nodes and impairing an HCMV-specific immune response [105].

The expression of the UL 111a gene, which encodes a functional IL-10 homolog with strong immunosuppressive effects, also offers HCMV strategy in viral latency to suppress the immune system. Through the latent infection process, UL111a undergoes alternative splicing, which results in the expression of a latency related transcript cmvIL-10, and the production of a protein that mimics the function of human immunosuppressive cytokine IL-10. This favors the infected cells not to be recognized by the immune system and to avoid clearance. Also, US28 and UL144 are expressed during latency redirect the immune response or block the immune recognition [107].

Latent HCMV infection also modulate the cell expression of MHC molecules class I in order to evade the immune response. At least four proteins encoded by (US) region of HCMV genome involved in inhibition of MHC class I expression, either by directly acting on MHC class I moleculeor acting on MHC class I-associated proteins, such as TAP (transporter associated with antigen processing) and tapas in [101]. These loci of HCMV DNA encodes several distinct IE proteins, pUL36, pUL37, and pUL37\_1, which appear to stop engagement of the apoptotic signals associated with the tumor necrosis factor (TNF) family of receptors including Fas. As a result, the activation of Fas-associated death domain (FADD) is inhibited, which prevents activation of procaspase-8 FLICE (FADD-like interleukin-1 beta-converting enzyme) and ultimately prevent the active caspase-8 to cause the subsequent activation of downstream effector caspases and prevent apoptosis of the cells [107, 108].

#### *1.3.3 Transmission*

The spreading of HCMV from one person to another primarily occurs through infected bodily secretions such as saliva, blood, tears, milk, and urine [109]. Close or intimate person to person contact usually is necessary for viral transmission. Accordingly, sexual transmission has been implicated in the spread of CMV. Seropositivity is higher among persons with multiple sexual partners and histories of sexually transmitted diseases [110].

Cytomegalovirus (CMV) may be transmited from mother to infant before, during or after birth. During pregnancy, vertical CMV transmission occurs via either passage of virions from maternal blood to the fetus and subsequent infection of the placenta or via the entry of infected maternal leukocytes, endometrial, or cervical cells into the fetal circulation. The infection ascending from the genital tract may also be possible. During birth, the infection of neonate occurs via the contact with infected maternal genital secretions. After birth, breastfeeding is the most frequent route of CMV transmission to the neonate [111]. Cytomegalovirus transmission also occurs naturally after receipt of an organ containing latent virus where these transplant recipients (who are undergoing immunosuppressive treatment) are at risk of contracting the disease [112].

#### *1.3.4 CMV clinical features*

#### *1.3.4.1 CMV infections in the immunocompetent host*

Cytomegalovirus (CMV) infection is common among patients of all age groups, but it has traditionally been considered as a problem in neonatal and immunosupressed patients. Cytomegalovirus infection in immunocompetent patients is usually asymptomatic or subclinical. Symptomatic disease usually results in mononucleosis-like syndrome. The symptoms are similar to classic mononucleosis, caused by the Epstein–Barr Virus. However, the mononucleosis syndrome

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*1.3.4.2 CMV infections in the immunocompromised patients*

in 1970 [114].

HIV-infected patients [115].

neurological diseases [120].

economically depressed regions) **Figure 10** [122].

*1.3.5 Epidemiology*

*1.3.4.3 Congenital infection and diseases*

associated with CMV typically lacks signs of enlarged cervical lymph nodes and splenomegaly [113]. Rarely, CMV infection in immunocompetent persons is associated with severe diseases such as enteritis, thrombotic disease, hemolytic anemia, thrombocytopenia, encephalitis, Guillain–Barré syndrome and myocarditis.

Guillain–Barré syndrome is immune-mediated peripheral neuropathy characterized by neuromuscular paralysis. The first case of Guillain-Barré Syndrome associated with cytomegalovirus (CMV) infection was reported in a renal transplant recipient

CMV infection is one of the most important infectious complications of solidorgan transplantation and is responsible for serious, life-threatening diseases in patients infected with human immunodeficiency virus (HIV) and other immunodeficiencies [115]. CMV disease manifests in the vast majority of transplant recipients as a viral syndrome that includes fever, malaise, myalgia, or headache (sometimes called *CMV syndrome*) or more severe organ-specific diseases such as pneumonitis, gastrointestinal lesions, hepatitis, retinitis, pancreatitis, myocarditis, and rarely, encephalitis or peripheral neuropathy. In solid organ transplant (SOT) recipients, primary HCMV infection has been consistently linked with dysfunction of the transplanted organ. In HIV-infected patients, retinitis is the single most common manifestation of CMV disease, accounting for 85% of all cases [116]. In developing countries, CMV retinitis is still the most frequent cause of visual loss in

Congenital infection refers to a condition where cytomegalovirus is transmitted in the prenatal period. Worldwide, approximately 1 in 100 to 500 babies are born with congenital CMV. Approximately 1 in 3000 will show symptoms and 1 in 7000 will die [117]. Congenital HCMV infection occurs after primary infection (or reactivation) during pregnancy. Congenital infections are less common in poorer communities with high seropositivity of, women in childbearing age.. In industrialized countries, up to 8% of HCMV seronegative mothers acquire primary HCMV infection during pregnancy, of which roughly 50% will transmit CMV to the fetus [118]. Between 10–15% of infected fetuses are born with the symptoms of congenital CMV disease, [119] which may include pneumonia, gastrointestinal, retinal, and

Human CMV is an ancient virus that is ubiquitous in human populations, reaching a prevalence of 100% in Africa and Asia, and approximately 80% in Europe and the USA, depending on socioeconomic status [121]. Seroprevalence rates of HCMV vary depending on age (higher rates are observed among older persons), geography (higher rates in developing countries), and socioeconomic status (higher rates in

A comparison of literature estimates shows that congenital CMV-related disabilities are as common among newborns and children as other better known diseases such as Down syndrome, fetal alcohol syndrome, or spina bifida [123]. Of the approximately 30,000 United States babies born with an infection with CMV every year almost 20 percent are born with or experience permanent sequelae such

as hearing loss, eyesight loss, brain damage or cognitive impairment.

#### *Human Herpetic Viruses and Immune Profiles DOI: http://dx.doi.org/10.5772/intechopen.96340*

associated with CMV typically lacks signs of enlarged cervical lymph nodes and splenomegaly [113]. Rarely, CMV infection in immunocompetent persons is associated with severe diseases such as enteritis, thrombotic disease, hemolytic anemia, thrombocytopenia, encephalitis, Guillain–Barré syndrome and myocarditis. Guillain–Barré syndrome is immune-mediated peripheral neuropathy characterized by neuromuscular paralysis. The first case of Guillain-Barré Syndrome associated with cytomegalovirus (CMV) infection was reported in a renal transplant recipient in 1970 [114].

#### *1.3.4.2 CMV infections in the immunocompromised patients*

CMV infection is one of the most important infectious complications of solidorgan transplantation and is responsible for serious, life-threatening diseases in patients infected with human immunodeficiency virus (HIV) and other immunodeficiencies [115]. CMV disease manifests in the vast majority of transplant recipients as a viral syndrome that includes fever, malaise, myalgia, or headache (sometimes called *CMV syndrome*) or more severe organ-specific diseases such as pneumonitis, gastrointestinal lesions, hepatitis, retinitis, pancreatitis, myocarditis, and rarely, encephalitis or peripheral neuropathy. In solid organ transplant (SOT) recipients, primary HCMV infection has been consistently linked with dysfunction of the transplanted organ. In HIV-infected patients, retinitis is the single most common manifestation of CMV disease, accounting for 85% of all cases [116]. In developing countries, CMV retinitis is still the most frequent cause of visual loss in HIV-infected patients [115].

#### *1.3.4.3 Congenital infection and diseases*

Congenital infection refers to a condition where cytomegalovirus is transmitted in the prenatal period. Worldwide, approximately 1 in 100 to 500 babies are born with congenital CMV. Approximately 1 in 3000 will show symptoms and 1 in 7000 will die [117]. Congenital HCMV infection occurs after primary infection (or reactivation) during pregnancy. Congenital infections are less common in poorer communities with high seropositivity of, women in childbearing age.. In industrialized countries, up to 8% of HCMV seronegative mothers acquire primary HCMV infection during pregnancy, of which roughly 50% will transmit CMV to the fetus [118]. Between 10–15% of infected fetuses are born with the symptoms of congenital CMV disease, [119] which may include pneumonia, gastrointestinal, retinal, and neurological diseases [120].

#### *1.3.5 Epidemiology*

Human CMV is an ancient virus that is ubiquitous in human populations, reaching a prevalence of 100% in Africa and Asia, and approximately 80% in Europe and the USA, depending on socioeconomic status [121]. Seroprevalence rates of HCMV vary depending on age (higher rates are observed among older persons), geography (higher rates in developing countries), and socioeconomic status (higher rates in economically depressed regions) **Figure 10** [122].

A comparison of literature estimates shows that congenital CMV-related disabilities are as common among newborns and children as other better known diseases such as Down syndrome, fetal alcohol syndrome, or spina bifida [123]. Of the approximately 30,000 United States babies born with an infection with CMV every year almost 20 percent are born with or experience permanent sequelae such as hearing loss, eyesight loss, brain damage or cognitive impairment.

**Figure 10.**

*Globally, rates of CMV seroprevalence in women of reproductive age and the incidence of congenital CMV infection. Studies have been performed in Australia, Belgium, Brazil, Canada, Chile, England, Finland, France, Germany, Ghana, India, Israel, Italy, Japan, South Africa, Spain, Sweden, Taiwan, Turkey, and the USA.*

The contribution of congenital CMV infection to childhood hearing loss is of particular importance, with approximately 20% of moderate to profound bilateral sensorineural hearing impairment occurring due to congenital CMV infection [124]. Approximately 90% of newborns with congenital infection have no symptoms at birth; If symptoms are present, they are often nonspecific [125]. When disabilities such as hearing loss appear, often months or years later, it is usually too late to make a retrospective diagnosis that identifies congenital CMV infection as the cause. For an individual woman, the greatest risk of having a baby with congenital infection comes from the mother's primary infection during pregnancy [126]. Consequently, babies of women who are CMV negative prior to pregnancy are particularly vulnerable to poor outcomes if the mother becomes infected during pregnancy [127].

In a nationally representative survey it has been stated that between 30% and 50% of United States women under 45 years of age are seronegative for CMV and that as many as a half a million US women of childbearing age experience primary CMV infections every year [128].

#### *1.3.6 Immune evasion by Cytomegalovirus*

HCMV persistence is correlated with the interaction between the immune response of the host and the virus evasion mechanisms, where HCMV interferes with both adaptive immune responses and immune effectors. A variety of evasion strategies have been developed by HCMV to prevent selected dendritic cell functions. The differentiation of CMV infected monocyte into macrophages and CD1 a-positive Dendritic cells (DCs) is inhibited, which does not require viral replication [129].

#### *1.3.6.1 Evasion of Innate immunity*

The innate immune system is essential in driving an efficacious acquired immune response. This includes the induction of type I interferons, activation of professional antigen presenting cells, and recruitment and activation of NK cells which themselves promote more efficient activation of antigen presenting cells and T cells. The binding and entry of HCMV into the cell initiates several pathways leading to the upregulation of NFkB and interferon regulatory factor 3 (IRF3) which can ultimately lead to the production of type I interferons and certain

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ated NK cell activation [132].

*1.3.6.2 Evasion of adaptive immunity*

recognize the viral surface glycoproteins gB and gH [130].

*1.3.6.1.1 Natural Killer (NK) cells and immune evasion mechanisms*

inflammatory cytokines. This innate cellular response to the initial stages of infection is mediated by Toll like receptor 2 (TLR2) signaling, which has been shown to

The importance of NK in the innate immune response to HCMV is suggested by the extensive studies of mechanism of HCMV prevention of the activation of NK cells. NK cells are inhibited by signals sent via inhibitory receptors that interact with class I MHC molecules on the surface of target cell. Low surface levels of Class I MHC on HCMV-infected cells may also reduce the inhibitory signaling of NK. This could make the infected cells susceptible to NK cell cytotoxicity [131]. Two mechanisms describing HCMV-mediated inhibitory receptor signaling have been reported. Firstly, HCMV uses the host HLA-E pathway to inhibit NK cells via the CD94/NKG2 heterodimeric inhibitory receptor by promoting cell surface HLA-E expression. Viral UL40 protein contains a nonomeric peptide which binds HLA-E promoting its cell surface expression. Secondly, HCMV expresses a viral homologue of cellular MHC Class I, UL18. UL18 is trafficked to the cell surface where it binds to the inhibitory NK cell receptor, LILRB1 (LIR-1) with higher affinity than MHC Class I inhibiting LILRB1+ NK cell activation. HCMV encodes five genes controlling NK cell activation and cytotoxicity by the provision of inhibitory signals and suppression of activating signals. The pp65 tegument protein (UL83) dissociates CD3\_ signaling from NKp30, whilst intracellular retention of CD155 and CD112 by UL141 prevents activation of NK cells via receptors CD226 and CD96, the remaining viral proteins interfere with a major receptor on all human NK cells (NKG2D) that medi-

Primary infection of HCMV provokes the production of antibodies specific for many HCMV proteins including structural tegument proteins such as (pp65 and pp150), envelope glycoproteins predominantly (gB and gH) as well as non-

In human bone marrow transplantation studies where HCMV infection can cause significant morbidity, it was evident that there was a strong correlation between recovery of the CD8+ T cell population and protection from CMV disease. HCMV employs several mechanisms to interfere with the normal cellular MHC Class I processing and presentation pathways to prevent CD8+ T cell recognition. HCMV encodes at least four related glycoproteins, each with a unique mechanism to prevent antigen presentation. HCMV viral genes US2 and US11 degrade newly synthesized MHC class I heavy chains, US3 retains MHC-I in the endoplasmic reticulum by interfering with chaperone-controlled peptide loading and US6 inhibits the translocation of viral and host peptides across the endoplasmic reticulum membrane by the dedicated peptide transporter TAP (transporter associated with antigen processing) [133]. The action of these genes may not completely protect cells from CD8+ T cell recognition dependent on the T cell-antigen specificity. Whereas HCMV-infected cells expressing US2–11 prevent any presentation of IE antigen to human T cells, pp65 peptides were still presented. CMV-specific T cells are also marked by the lack of expression of the costimulatory receptors CD27 and

structural proteins such as the Immediate Early 1 protein (IE1, UL123).

CD28, which are otherwise commonly expressed on naïve T cells [134].

*1.3.6.2.1 CD8+ T cell responses and MHC Class I downregulation*

#### *Human Herpetic Viruses and Immune Profiles DOI: http://dx.doi.org/10.5772/intechopen.96340*

inflammatory cytokines. This innate cellular response to the initial stages of infection is mediated by Toll like receptor 2 (TLR2) signaling, which has been shown to recognize the viral surface glycoproteins gB and gH [130].

#### *1.3.6.1.1 Natural Killer (NK) cells and immune evasion mechanisms*

The importance of NK in the innate immune response to HCMV is suggested by the extensive studies of mechanism of HCMV prevention of the activation of NK cells. NK cells are inhibited by signals sent via inhibitory receptors that interact with class I MHC molecules on the surface of target cell. Low surface levels of Class I MHC on HCMV-infected cells may also reduce the inhibitory signaling of NK. This could make the infected cells susceptible to NK cell cytotoxicity [131]. Two mechanisms describing HCMV-mediated inhibitory receptor signaling have been reported. Firstly, HCMV uses the host HLA-E pathway to inhibit NK cells via the CD94/NKG2 heterodimeric inhibitory receptor by promoting cell surface HLA-E expression. Viral UL40 protein contains a nonomeric peptide which binds HLA-E promoting its cell surface expression. Secondly, HCMV expresses a viral homologue of cellular MHC Class I, UL18. UL18 is trafficked to the cell surface where it binds to the inhibitory NK cell receptor, LILRB1 (LIR-1) with higher affinity than MHC Class I inhibiting LILRB1+ NK cell activation. HCMV encodes five genes controlling NK cell activation and cytotoxicity by the provision of inhibitory signals and suppression of activating signals. The pp65 tegument protein (UL83) dissociates CD3\_ signaling from NKp30, whilst intracellular retention of CD155 and CD112 by UL141 prevents activation of NK cells via receptors CD226 and CD96, the remaining viral proteins interfere with a major receptor on all human NK cells (NKG2D) that mediated NK cell activation [132].

#### *1.3.6.2 Evasion of adaptive immunity*

Primary infection of HCMV provokes the production of antibodies specific for many HCMV proteins including structural tegument proteins such as (pp65 and pp150), envelope glycoproteins predominantly (gB and gH) as well as nonstructural proteins such as the Immediate Early 1 protein (IE1, UL123).

#### *1.3.6.2.1 CD8+ T cell responses and MHC Class I downregulation*

In human bone marrow transplantation studies where HCMV infection can cause significant morbidity, it was evident that there was a strong correlation between recovery of the CD8+ T cell population and protection from CMV disease. HCMV employs several mechanisms to interfere with the normal cellular MHC Class I processing and presentation pathways to prevent CD8+ T cell recognition. HCMV encodes at least four related glycoproteins, each with a unique mechanism to prevent antigen presentation. HCMV viral genes US2 and US11 degrade newly synthesized MHC class I heavy chains, US3 retains MHC-I in the endoplasmic reticulum by interfering with chaperone-controlled peptide loading and US6 inhibits the translocation of viral and host peptides across the endoplasmic reticulum membrane by the dedicated peptide transporter TAP (transporter associated with antigen processing) [133]. The action of these genes may not completely protect cells from CD8+ T cell recognition dependent on the T cell-antigen specificity. Whereas HCMV-infected cells expressing US2–11 prevent any presentation of IE antigen to human T cells, pp65 peptides were still presented. CMV-specific T cells are also marked by the lack of expression of the costimulatory receptors CD27 and CD28, which are otherwise commonly expressed on naïve T cells [134].

#### *1.3.6.2.2 CD4+ T cell responses and MHC Class II downregulation*

During primary infection, HCMV specific CD4+ T cells can be detected 7 days after the detection of HCMV DNA in peripheral blood in response to same ORFs asCD8 + T cells and pp65 and IE genes gB and gH. These cells produce T helper type 1 (Th1) cytokines IFNγand TNF. A large number of HCMV- encoded gene products target the MHC class I antigen presentation pathway in an effort to avoid recognition by CD8+ T cell [134].

Human cytomegalovirus also avoids the CD4+ T cell response by several ways. Disrupting IFN- induced, upregulation of MHC class II molecules to the cell surfaces by preventing the expression of Janus kinase 1 and suppression of Class II transactivator mRNA. The virally-encoded gene product of US2 also suppress MHC class II presentation to CD4+ T cells by redirecting the HLA-DR-and HLA-DMchains to the cytosol where they are degraded. HCMV re-programs human hematopoietic progenitor cells (HPCs) into immune-suppressive monocytes that express IL-10 in a process requiring US28. Recently a truncated transcript to UL111A, a viral homolog of the immunomodulatory cytokine IL-10, which is expressed during latency (cmvLA IL-10) has been shown to downregulate expression of MHC class I and class II molecules, inhibit both proliferation of mononuclear cells and the production of inflammatory cytokines [135].

#### *1.3.7 CMV prevention and vaccines*

The lack of specific and effective treatments for HCMV infection has highlighted the need to understand HCMV host-cell interactions, including viral entry and host immune responses against this virus [136]. The HCMV vaccine is designed to be used to prevent infection or to prevent its re-activation in those infected already [137]. To eliminate and eradicate congenital HCMV infection, vaccination would be a priority. Several researchers have attempted to develop vaccines against HCMV, such as live attenuated vaccines, recombinant chimeric vaccines, and subunit vaccines such as glycoprotein B and tegument protein pp65 vaccines [138]. However, none of these vaccines exhibited effective protection, as well as, and to this day, no licensed HCMV vaccine is available.

Strategies to reduce Congenital CMV disease burden may be implemented at different stages, and include prevention of maternal infection, prevention of motherto-child transmission, early detection and intervention by neonatal screening, and neonatal antiviral therapy. The principal sources of exposure for women of childbearing age are sexual contacts and children secreting the virus [136]. Pregnant women also should be advised to avoid close contact with individuals likely to shed CMV such as adults with symptoms consistent with mononucleosis and toddlers attending group day care. Preemptive therapy is an approach in which patients are monitored for early replication (ie, viremia by polymerase chain reaction [PCR]), and the antiviral drug is administered only when CMV replication is detected to prevent its progression to higher-grade viremia and CMV disease. Small, noncomparative studies have shown lower rates of CMV disease (especially delayed-onset disease) with preemptive compared with the use of antiviral prophylaxis in CMV-seronegative liver transplant recipients with seropositive donors [139].

**181**

**Author details**

Marwa Mohammed Ali Jassim1

and Murtada Hafedh Hussein3

, Majid Mohammed Mahmood<sup>2</sup>

1 Studies and Planning Department, University of Baghdad, Baghdad, Iraq

© 2021 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,

2 College of Science, Mustansiriyah University, Baghdad, Iraq

\*Address all correspondence to: majidmahmood93@yahoo.com

3 College of Science, Thi-Qar University, Thi-Qar, Iraq

provided the original work is properly cited.

\*

*Human Herpetic Viruses and Immune Profiles DOI: http://dx.doi.org/10.5772/intechopen.96340* *Human Herpetic Viruses and Immune Profiles DOI: http://dx.doi.org/10.5772/intechopen.96340*

## **Author details**

Marwa Mohammed Ali Jassim1 , Majid Mohammed Mahmood<sup>2</sup> \* and Murtada Hafedh Hussein3

1 Studies and Planning Department, University of Baghdad, Baghdad, Iraq

2 College of Science, Mustansiriyah University, Baghdad, Iraq

3 College of Science, Thi-Qar University, Thi-Qar, Iraq

\*Address all correspondence to: majidmahmood93@yahoo.com

© 2021 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.

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**191**

**Chapter 7**

**Abstract**

disease outcome

**1. Introduction**

to Thymus

Plasticity in Interferon Responses

Parasitic infections are the major threat prevalent in tropical and subtropical regions throughout the world. Different parasitic infections take a huge toll on mortality and morbidity at global level. Different parasites invade the host system, multiply inside host cells of their choice and sabotage defense mechanisms to overpower the host. T-cell immunity is majorly affected in different parasitic diseases such that the peripheral T-cell immune response is altered along with lesser explored thymic changes. Direct and/or indirect effect of parasitic infection leads to alterations in T-cell development, differentiation and activation resulting in deregulated T-cell immune mechanisms. Cytokines of interferon family play a significant role in determining the disease outcome and severity. Therefore, in this chapter, we here provide a detailed overview of the functional role played by IFNs during parasitic diseases in terms of their influence on peripheral T-cell activation and tolerance along with lesser explored impact on developing T cells in the thymus

**Keywords:** parasitic diseases, periphery, IFN, T cells, thymus, immunomodulation,

Parasitism is a relationship among species, in which one organism, the parasite, sustains on the host organism. Parasitic diseases can affect almost all living organisms. Parasites are dependent on the host organisms for their own survival. Not all parasites are harmful but some cause severe pathology to the host, such as *Leishmania*, *Plasmodium*, *Trypanosoma*, etc. Parasites known to affect humans are divided into three classes: protozoans, helminths and ectoparasites [1]. Parasite invasion triggers the innate, inflammatory and adaptive immune responses inside the mammalian host. Innate immunity recognizes the non-self and activates the T-cell–mediated adaptive immune system in order to eliminate the invader. Removal or recruitment of parasite is dependent on the production of distinct pattern of cytokines from specific T cells. T cells are formed through an intricate

Modulates T-Cell Immunity in

Parasitic Infections: Periphery

*Lovlesh Thakur, Nadeem Akhtar, Aklank Jain,* 

*Hridayesh Parkash and Manju Jain*

with altered microenvironmental niches.

#### **Chapter 7**
