**3. TLR signaling pathway**

*Toll-like Receptors*

**2. Cells expressing TLRs**

Among the two types of vertebrate immune system, innate immunity provides the first line of defense against parasites. Previous studies stated innate immunity as nonspecific response, and it induces the acquired immunity (slower and specific response) by providing pathogens to T and B cells [5]. However, recent evidence proved that innate immune system also had a great degree of specificity and can provide host defense against invading parasites. This is because of the presence of five classes of pattern recognition receptors: TLRs (Toll-like receptors), C-type lectin receptors, NOD-like receptors (nucleotide-binding oligomerization domain leucine-rich repeat-containing receptors), RIG-I (retinoic acid inducible gene I protein) helicase receptors, and cytosolic dsDNA sensors [6, 7]. Among them, TLRs form a bridge between innate and adaptive immunity and play a very important role in parasite eradication. TLRs recognize specific pathogen-associated molecular patterns (PAMPs) in pathogens and initiate opsonization, phagocytosis, pro-

inflammatory and anti-inflammatory response, and apoptosis [7, 8].

[17]. **Table 1** shows the distribution of various TLRs in different cells.

TLRs can be classified on the basis of their recognized ligands—TLR1/TLR2 heterodimer (triacylated lipopeptides), TLR2/TLR6 heterodimer (diacylated lipopeptides), TLR4 (lipopolysaccharide), TLR3 (double-stranded RNA), TLR5

**Cells Expressing TLRs** Neutrophils TLR 1, 2, 4, 5, 6, 7, 8 Monocytes/macrophages TLR 1, 2, 4, 5, 6, 7, 8 Myeloid dendritic cells TLR 2, 3, 4, 7, 8 Plasmacytoid dendritic cells (PDCs) TLR 1, 6, 7, 9 B lymphocytes TLR 1, 3, 6, 7, 9, 10 T lymphocytes (Th1/Th2) TLR 2, 3, 5, 9 T lymphocytes (regulatory) TLR 2, 5, 8 Peripheral blood mononuclear cell (PBMC) TLR 2, 4, 5, 7, 8, 9

TLRs, a major component of innate immunity, are Type-1 transmembrane glycoproteins present in both vertebrates and invertebrates [9]. Toll-like receptors are named due to their similarity with Drosophila Toll protein (Toll) [10]. All TLRs have a highly variable extracellular domain containing leucine-rich repeat (LRR) domain for ligand binding and intracellular TIR homology domain [11]. Toll-like receptors and interleukin-1 receptor together form "Interleukin-1 receptor/Toll-like receptor" superfamily whose all members have a common Toll-IL-1 receptor (TIR) domain [12]. Till date, 10 humans and 12 mice functional TLRs were identified. Although humans and mice have similar TLR1–9, TLR10 is nonfunctional in mice and TLR11–13 are lost in humans [13]. TLR1, TLR2, TLR4, TLR5, and TLR6 recognize extracellular PAMPs, which are expressed on cell surface, whereas TLR3, TLR7, TLR8, and TLR9 are expressed within endoplasmic reticulum (ER), endosomes, lysosomes, and endolysosomes and identify nucleic acids [14]. The presence of TLRs on specific intracellular vesicles restricts their activation by self-nucleic acids released by apoptotic cells [15]. TLR11 (a relative of TLR5) and TLR13 are expressed in intracellular vesicles [16], but cognate PAMP of TLR13 has not been identified yet

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**Table 1.**

*Different Toll-like receptors expressed by immune cells [7, 16].*

TLRs present on dendritic cells (DCs) [both myeloid DCs (mDCs) and plasmacytoid DCs (pDCs)], neutrophils, macrophages, natural killer (NK), and natural killer T (NKT) cells induce dendritic cell maturation, MHC molecule upregulation, and costimulatory molecule production (CD40, CD80, and CD86) [21, 22]. The cytokines released by TLR signaling ultimately activate Th1 cells (via IL-12 from DCs) and Th2 cells (via IL-4 from B cell) [21, 23].

Toll-interleukin-1 receptor (TIR) domain is responsible for transducing the signal from TLRs to their adaptor proteins. The C-terminus of all TLRs, IL-1 and IL-18 and adaptor proteins of TLRs have this TIR domain. Six adaptor proteins involved in TLR signaling are MyD88 (myeloid differentiation factor 88), TIRAP (Toll-IL-1 receptor domain-containing adaptor protein) and MAL (MyD88 adapter-like), TRIF (TIR domain-containing adaptor inducing interferon-β) and TICAM-1, TRAM (TRIFrelated adaptor protein) and TICAM-2, SARM (sterile-α and HEAT/Armadillo motifs-containing protein) and MyD88-5, and BCAP (B Cell Adaptor for PI3K) [24]. TLR signaling occurs via two separate pathways: MyD88 (myeloid differentiation primary response protein)-dependent pathway and MyD88-independent pathway. MyD88-dependent pathway stimulates all TLRs except TLR-3, which gets stimulated by MyD88-independent pathway. However, in case of TLR4, both MyD88-dependent and independent pathways operate [25]. MyD88 (an adaptor molecule) activates IRAK-4 (interleukin-1 receptor-associated kinase-4) alone or in combination with TIRAP (Toll-IL-1 receptor domain-containing adaptor protein) or MAL (MyD88

**Figure 1.** *TLR signaling pathway.*

adapter-like). Then, IRAK-4 phosphorylates IRAK-1 [26] which in turn phosphorylates IRAK-2. IRAK-2 ubiquitinates TRAF6 (tumor necrosis factor receptorassociated factor 6) and induces two signaling pathways: (1) AP-1 (activator protein 1) activation via MAK 4/7 (mitogen-activated protein kinase) phosphorylation and (2) TAK1 (transforming growth factor-β-activated kinase 1) activation ultimately leads to MAPK (mitogen-activated protein kinase) and IKK complex [27] stimulation and nuclear factor κB (NF-κB) translocation inside the nucleus via degradation of its inhibitor. Both AP-1 and NF-κB induce the expression of pro-inflammatory cytokines and chemokines. A different MyD88-dependent pathway stimulates TLR 7, 8, and 9, which acts as a ligand for viral nucleic acids. MyD88-associated IRAK1 (interleukin-1 receptor-associated kinase-1) phosphorylates IRF7 (interferonregulatory factor-7), which regulates Type I interferon expression [28]. TLR signaling through MyD88-independent pathway occurs via two adaptor molecules—TRIF (Toll-IL-1 receptor domain-containing adaptor inducing interferon-β) and TRAM (TRIF-related adaptor molecules) (**Figure 1**). This induces Type 1 interferon by IRF-3 (interferon-regulatory factor-3), NF-κB activation, and expression of costimulatory molecules [29].
