Toll-Like Receptors in Immunity and Inflammation

Abstract

Toll-like receptors (TLRs) are critical components of innate immunity and serve as pattern recognition receptors (PRRs). These PRRs recognize different microbe or pathogen-associated molecular patterns (MAMPs or PAMPs) and death/danger-associated molecular patterns to initiate the pro-inflammatory immune reaction in response to foreign and internal dangers. PRRs, including TLRs, also connects innate immunity to adaptive immunity. Furthermore, TLRs expressed on both innate and adaptive (T and B cells) immune cells regulate their functions. TLRs were first discovered in the common fruit fly or Drosophila melanogaster as genes controlling dorso-ventral body patterning during embryonic development. Immunological and scientific advances have led to the discovery of different TLRs (extra and intracellular) with diverse functions. The present chapter introduces the role of TLRs in immunity and inflammation and their expansion to mammalian reproduction and embryonic development, maintenance of immune homeostasis, health, and disease, specifically neurological disorders, including neurodegeneration and cancers.

1. Introduction

The discovery of toll-like receptors (TLRs) revolutionized the field of immunology by filling the gap that the immune system recognizes and clears pathogens to maintain immunohomeostasis or immune homeostasis. For example, the first discovery of TLR4 in human spleen, intestinal epithelial cells (IECs), and peripheral blood leukocytes (PBLs), including monocytes, macrophages, dendritic cells (DCs), T and B cells, and its downstream signaling via NF-κB upon recognizing the corresponding pathogen/microbe-associated molecular patterns (PAMPs or MAMPs) such as lipopolysaccharide (LPS) showed its involvement in the activation and regulation of the adaptive immunity [1, 2, 3]. This groundbreaking research led to the reemergence of innate immunity in mainstream immunology research and the subsequent discovery of many other TLRs. For example, 13 TLRs in laboratory mice (TLR1-TLR13) and 10 TLRs in humans (TLR1-TLR10) recognizing different ligands are known today (Table 1) [2, 5].

TLRs	TLR Localization	Ligands (PAMPs and DAMPs)	Origin of Ligands
TLR1	Plasma membrane	Triacyl lipopeptide soluble factors	Bacteria and mycobacteria
TLR2	Plasma membrane and endosomes	Peptidoglycan (PGN), lipoteichoic acid (LTA), Lipoproteins or lipopeptides, lipoarabinomannan, glycolipids, porins, zymosan, atypical LPS, heat shock protein 70 (Hsp70), eosinophil-derived neurotoxin (EDN) acts an alarmin	Gram +ve bacteria, mycobacteria, S. epidermidis, Trypanosoma cruzi, Treponema maltophilum, Neisseria, Fungi, Leptospira interrogans, Porphyromonas gingivalis, host
TLR3	Endoplasmic reticulum (ER), endosomes, multivesicular bodies, lysosomes, and Endolysosomes	dsRNA and ssRNA and synthetic analog polyinosinic-polycytidylic acid (poly I:C)	dsRNA, ssRNA, and dsDNA Viruses
TLR4	Plasma membrane and endosome	LPS, Taxol, Fusion protein, Envelope proteins, high mobility group box 1 protein (HMG-B1), Hsp60, Hsp70, Hsp22, Hsp96 Type III repeat extra domain A of fibronectin, hyaluronic acid, heparin sulfate, Fibrinogen, Saturated fatty-acids and Fetuin-A	Gram negative bacteria, Plant, respiratory syncytial virus (RSV), mouse mammary tumor virus (MMTV), Chlamydia pneumoniae, Chlamydia trachomatis, host
TLR5	Plasma membrane	Flagellin	Bacteria
TLR6	Plasma membrane	Di-acyl lipopeptides, Zymosan	Mycoplasma
TLR7	ER, endosomes, multivesicular bodies, lysosomes, and Endolysosomes	guanosine- and uridine-rich ssRNA, Loxoribine (a guanine analog), Bropirimine, ribonucleoproteins (RNPs), siRNAs, and imidazoquinoline derivatives such as resiquimod (R848) and imiquimod	Viruses (human immunodeficiency virus-1 or HIV-1, Influenza virus and vesicular stomatitis virus or VSV), synthetic compounds
TLR8	ER, endosomes, multivesicular bodies, lysosomes, and Endolysosomes	ssRNA, RNPs	Viruses
TLR9	ER, endosomes, multivesicular bodies, lysosomes, and Endolysosomes	CpG oligodeoxyneucleotide (ODN), viral double stranded DNA (dsDNA), Hemozoin pigment	Bacteria and viruses (Herpes Simplex Virus-1 and -2 or HSV-1 and -2, mouse cytomegalovirus or MCMV), Malaria, and protozoa genome (T. cruzi)
TLR10 (Humans)	ER, endosomes, multivesicular bodies, lysosomes, and Endolysosomes	dsRNA [4]	Viruses
TLR11	ER, endosomes, multivesicular bodies, lysosomes, and Endolysosomes	Profilin-like protein	Toxoplasma gondii
TLR12	ER, endosomes, multivesicular bodies, lysosomes, and Endolysosomes	Profilin-like protein	Toxoplasma gondii
TLR13 (Mouse)	ER, endosomes, multivesicular bodies, lysosomes, and Endolysosomes	23 s ribosomal RNA	Bacteria

Table 1.

Different TLRs in laboratory mice and humans, their localization, ligands, and their origin.

TLRs are expressed extracellularly (on the plasma membrane, TLR1, TLR2, TLR4, TLR5, TLR6, TLR10 (in humans) and TLR11, and TLR12 in mice) and intracellularly in endosomes, endolysosomes, and lysosomes (TLR3, TLR7, TLR8, and TLR9) (Table 1) [5, 6]. Mouse TLR13 resides in endosomes, but its ligand is unknown [6, 7]. However, some immune cells, such as dendritic cells (DCs), epithelial cells, and endothelial cells, also have intracellular TLR2 and TLR4 [8, 9, 10]. TLRs are members of the pattern recognition receptor (PRR) family, which frequently recognize molecules expressed and released by microbes and damaged or dying cells, called damage/death-associated molecular patterns (DAMPs), to initiate pro-inflammatory immune response for their clearance. The clearance of the invading agent proceeds to the resolution phase of the inflammation for maintaining immune homeostasis that otherwise activates the adaptive immune system later. The combined protective action of innate and adaptive immunity fights back to take care of the outer or endogenous threat, failure of which leads to chronic inflammatory diseases and many cancers.

It is important to note that TLR homolog (called Toll) was first discovered in the Drosophila melanogaster (D. melanogaster) or fruit fly as a gene responsible for dorsoventral body patterning during embryonic development in 1980 [3, 11]. Hence, the story of TLRs’ discovery is fascinating, starting with embryonic development and ending with their immunological functions and immune homeostasis maintenance. The current introductory chapter discusses TLRs in the context of human immunity, their impact on immune-mediated diseases, and their use in drug discovery and immunotherapeutic approaches. We will not discuss the myeloid differentiation primary-response protein 88 (MyD88)-dependent and -independent or TIR-domain-containing adapter-inducing interferon-β (TRIF) or TIR-domain-containing adapter molecule 1 (TICAM-1)-dependent TLR signaling pathways activating NF-κB and interferon regulatory factors (IRFs)-dependent cytokines, chemokines, and type 1 interferons (IFNs) to initiate pro-inflammatory immune response as it is discussed elsewhere in detail [6, 12, 13, 14, 15, 16]. TIR-domain-containing adaptor protein (TIRAP) and TRIF-related adaptor molecule (TRAM) are other TIR-domain-containing adaptor molecules involved in MyD88 and TRIF-dependent signaling pathways [16]. However, we have included Figure 1 for MyD88- and TRIF-dependent TLR signaling activating NF-κB- and IRF-3 and IRF-7-mediated inflammatory immune response.

Figure 1.

TLR (MyD88-dependent and -independent or TRIF-dependent) signaling. The recognition of Gram-negative bacteria or LPS by TLR4 leads to the activation of downstream signaling pathways through the activation MyD88-dependent and -independent (TRIF-dependent) manner to activate NF-κB causing the transcription and translation of pro-inflammatory genes (cytokines and chemokines) and well as the generation of type 1 IFNs. The TRIF and MyD88-depedent signaling pathways downstream to TLR4 activation converge at transforming growth factor (TGF)-β-activated kinase 1 (TAB1), 2, and/or 3 and transforming growth factor beta-activated kinase 1 (TAK1) complex. TAK1 activation stimulates MAPK3/6 that activates AP1 and CREB along with activating NF-κB signaling via NF-κB essential modulator (NEMO). Activation of intracellular TLRs (TLR3, TLR7, TLR8, and TLR9) induces the generation of type 1 interferons (IFNs) that regulate immune response, including adaptive immunity. The activation TLR3 signaling via TRIF-dependent signaling activates TNAK-binding kinase 1 (TBK1) and IKK-1 activates interferon regulatory factor 3 (IRF3) and IRF7 to produce type 1 IFNs. On the other hand, TLR7/8 activation involves MyD88-dependent downstream signaling activating TRAF6 and IRAK4 complex formation that further activates IRAK1/ TNF receptor associated factor 3 (TRAF3)/osteopontin (OPN) complex formation to phosphorylate IRF7, which enters to the nucleus for inducing IRF7-dependent type 1 IFN production. Thus, TLR activation induces NF-κB and IRF-dependent pro-inflammatory immune response to clear the external or internal danger, but its overactivation causes exaggerated inflammation and predisposition to several inflammatory conditions, such as sepsis, cytokine storm, autoimmunity, and cancers.

2. TLRs are critical to maintain immune homeostasis

Homeostasis maintenance and immunological well-being, called immune homeostasis, is critical for healthy life and longevity. For example, TLR-mediated commensal microbes’ recognition maintains intestinal epithelial homeostasis and protects the host from gut injury and associated mortality [17]. Furthermore, TLR3-mediated macrophage priming for subsequent TLR7 activation involves the Janus-associated kinase (JAK) and signal transducer and activator of transcription (STAT) pathway activation that controls synergistic production of cytokines, innate immune memory generation, and immune homeostasis maintenance during temporally separated subsequent infection [18]. Thus, TLR3 and TLR7 crosstalk are critical for immune homeostasis and innate immune memory generation.

Immune homeostasis disruption causes several auto-immune, auto-inflammatory, and immunodeficiency diseases. Furthermore, foreign invasion from microbes, allergens, and xenobiotics disrupts the immune homeostasis by activating different PRRs, including TLRs, which becomes detrimental to the host once it persists longer. For example, several pro-inflammatory diseases, including sepsis-associated cytokine storm, coronavirus disease 2019 (COVID-19), and neuroinflammation involve TLR overactivation [5, 12, 13, 19].

Several endogenously expressed TLR signaling negative regulators (Table 2) prevent exaggerated TLR signaling responsible for inflammation and inflammatory diseases. The details of endogenous negative regulators of TLR signaling are beyond the scope of this introductory chapter and are discussed elsewhere [13, 16, 20, 21, 22, 23]. Hence, dysregulated TLR signaling in response to external and endogenous threats (PAMPs, MAMPs, or DAMPs) impairs immune homeostasis, causing cytokine syndrome or cytokine release syndrome (CRS) seen during sepsis, acute or severe COVID-19, and other inflammatory conditions [13, 24, 25]. In addition to overactivated TLR signaling-induced inflammatory conditions, the deficiency of TLR signaling molecules causes different primary immunodeficiency diseases (PIDs) [26, 27]. For example, TLR3 signaling defect correlates well with herpes simplex virus-1 (HSV-1) encephalitis, and a dampened TLR2 and TLR4 signaling has been observed in chronic granulomatosis disease (CGD) and X-linked agammaglobulinemia (XLA) [26, 27]. Table 3 shows different TLRs and their signaling pathway molecules’ deficiency and impact on immunity or PIDs. Therefore, balanced TLR signaling is critical for immune regulation or immune homeostasis [14, 25, 28, 29, 30, 31, 32, 33, 34].

Endogenous TLR signaling negative regulators	Classification	Mode of action
TRAM (Translocating chain-associated membrane protein) adaptor with Golgi dynamics (GOLD) domain) or TAG	Splice variant of TRAM	Competes with TRAM for binding to TRIF
Sterile alpha- and armadillo-motif-containing protein or SARM	TIR-domain containing adaptor molecule	Binds and inhibits TRIF-dependent TLR signaling
Interferon regulatory factor 4 or IRF4	IRF family of transcription factors	Inhibits IRF5 binding to MyD88 and TRAF6 and its translocation to nucleus
Tumor necrosis factor-α-induced protein 8-like 2 or TIPE2	Member of TNF-α-induced protein-8 (TNFAIP8) family	Inhibits TLR signaling via binding to CASP8 and inhibiting NF-κB and AP-1 activation
Bruton’s tyrosine kinase or Btk	Tec family tyrosine kinase	Phosphorylates MAL that activates SOCS-1
DNAX activation protein of 12 kDa or DAP12, also called KARAP	Transmembrane adaptor protein	DAP12 mediated inhibition of TLR signaling involves another adaptor protein called DOK-3
Downstream of kinase 1 and 2 (DOK1 and DOK2)	Adaptor Proteins	Inhibit Ras-Erk dependent signaling
Axl	TAM family of receptor kinases	Inhibits NF-κB mediated production of TNF-α
Interlukin-1 receptor-associated kinase-M (IRAK-M) or IRAK-3	Member of serine/threonine kinase family	Inhibits dissociation of IRAK-1 and IRAK-4 form MyD88 and formation of IRAK-TRAF6 complex
TOLL-interacting protein (TOLLIP)	Adaptor protein that interacts with cytoplasmic TIR domain of IL-1Rs	Inhibits phosphorylation and kinase activity of IRAK1
Src homology 2 domain-containing protein tyrosine phosphatase-1 (SHP-1)	Intracellular tyrosine phosphatase	Inhibits MAP kinase and NF-κB activation
Calcineurin	A serine/threonine phosphatase	Specific pathway unknown
Protein tyrosine phosphatase-1 B (PTP1B)	Intracellular tyrosine phosphatase	Inhibits MAPKs, NF-κB and IRF3
A20 or TNF-α-induced protein 3 (TNFAIP3)	Ubiquitin modifying enzyme	Inhibits NF-κB signaling as negative feedback by removing ubiquitin moieties from TRAF6
Cylindromatosis or CYLD	Tumor suppressor deubiquitinase	Inhibits TLR2 signaling via inhibiting MyD88, TRAF2, TRAF6 TRAF7 and NEMO
Ubiquitin-specific protease 4 or USP4	Deubiquitinase	Inhibits TRL4 signaling via deubiquinating TRAF6 and inhibiting its adaptor function
USP18 or UBP43 or ISG15 isopeptidase	Isopeptidase	It cleaves the K63-linked polyubiquitin chains of TAK1 and also targets NEMO
Deubiquitinating enzyme A (DUBA)	Cysteine protease	Removes K63-ubiquitin chain from TRAF3 to inhibit NF-κB and IRF3 activation
Nuclear receptor 4A1 or NR4A1 (Nur77)	Member of nuclear receptor 4A receptor subfamily	Prevent auto-ubiquitination of TRAF6 via binding to TRAF6
NR4A2 or Nurr1	Member of nuclear receptor 4A receptor subfamily	Inhibits NF-κB activation downstream to TLR4 signaling
Small heterodimer partner (SHP) or NR0B2	Orphan nuclear receptor	Prevents Lys63-linked polyubiquitination of TRAF6 and subsequent activation of NF-κB
Mitogen and stress-activated kinase 1 and 2 (MSK1 and 2)	Nuclear kinase sharing homology with ribosomal S6 kinase (p90^rsk) family	Phosphorylate histone H3 and CREB that negatively regulates TLR signaling and induces several anti-inflammatory genes
TRAF-associated NF-κB activator (TANK)	TRAF binding protein	Binding of TANK to TRAF6 inhibits NF-κB and AP-1 activation
PDZ and LIM domain containing protein or PDLIM-2 (Mystique and SLIM in mice)	PDZ and LIM domain containing Alkaline Phosphatase	Suppresses TLR signaling by acting as a nuclear E3 ubiquitin ligase and inhibits NF-κB activation
Mankorin ring finger protein 2 (MKRN2 or RNF62)	Zinc finger and RING finger domain containing nuclear protein	Binds to PDLIM2 and inhibits activation of NF-κB downstream to TLR signaling
PDLIM1 or CLP36 or Elfin	PDZ and LIM domain containing protein of APL subfamily	PDLIM1 inhibits NF-κB activation by sequestering p65 into cytosol
Tripartite motif-containing protein 30 A (TRIM30α)	A member of tripartite-motif (TRIM) protein family	Blocks TRAF6 autoubiquitination by degrading TAB2 and TAB3and suppresses NF-κB activation
TRIM8	Acts as ubiquitin E3 ligase	Polyubiquitinates TRF and inhibits TRIF-TBK1 interaction
Triad3A or Ring finger protein 216 (RNF216)	RING finger type E3 ubiquitin ligase	Degrades TLR proteins
NOD-like receptor family member X1 (NLRX1)	Member of NLR family	NLRX1 binds to IKK complex causing an inhibition of IKKα and IKKβ phosphorylation and NF-κB activation
NLRC3 or NOD3	Member of NLR family	NLRC3 interacts with TRAF6 to attenuate its K63-linked ubiquitination to inhibit NF-κB activation
NLRC5	Member of NLR family	Blocks IKK complexes to inhibit NF-κB activation and type 1 IFN signaling pathways
Serum stimulation 2 factor (ST2)	Serves as a part of IL-33 receptor	ST2 binds to sequesters MyD88 and MAL without affecting TRIF and IRAK to inhibit TLR-induced NF-κB activation
Single immunoglobulin IL-1R-relate receptor (SIGIRR or TIR-8)	Member of TLR/IL-1R superfamily	Blocks TLR-mediated NF-κB and JNK activation via stopping the recruitment of IRAK and TRAF6 towards MyD88
TLR10	Member of TLR family	Stimulates PI3K/Akt/IL-1R antagonist pathway and inhibits MyD88 and TRIF-dependent signaling pathways
Activating transcription factor 3 (ATF3)	Member of activating transcription factor/cAMP response element family of bZip transcription factors	Bind to consensus c-AMP response element (CRE) sequences
Interleukin-37 or IL-37	Member of IL-1 cytokine family	Blocks NF-κB and MAPK activation

Table 2.

Endogenous negative regulators of TLR signaling.

PIDs	Impact on TLR functions on immune cells
Common variable immunodeficiency (CVID)	Reduced expression and function of TLR7 and 9 on B cells, reduced activity of TLR7 and 9 on plasmacytoid DCs (pDCs)
Chronic granulomatous disease (CGD)	Increased TL 4 activity (increased TNF-α and IL-18 production) on neutrophils, peripheral blood monocytes (PBMCs), decreased TLR5 and TLR9 activity, TL 9 activity on B cells reduces as indicated by a decreased memory response
X-linked agammaglobulinemia (XLA)	Decreased TLR2 and 4 responses by monocytes and macrophages, monocyte-derived DCs show decreased TLR2, 4, 3, and 7/8 responses, including decreased DC maturation
Hyper Immunoglobulin E (IgE) syndrome	TLR2 and 4 response increases in PBMCs as indicated by increased TNF-α and IL-12 production
Adenosine deaminase (ADA) deficiency	Decreased TLR7 and 9-meidated immune response by B cells
Interleukin-receptor-associated kinase 4 (IRAK4) deficiency	Abolishes TLR7, 8, and 9-mediated type 1 IFN production, leaving TLR3 and TLR4-dependent IFN production intact

Table 3.

TLRs in different PIDs.

3. TLRs in human health, including embryonic development and disease

TLRs are responsible for dorso-ventral body patterning during the embryonic development of D. melanogaster. TLRs’ role in mammalian development is also emerging, for example, TLR 4 and 7 expression has been observed during murine embryonic development, and TLR7 expression has been observed in embryonic day 12 (E12) in the dorsal root ganglia (DRG) and nodose ganglion [35]. At E14, sympathetic ganglia, vagus nerve, and nerve fibers and ganglia in the respiratory apparatus express TLR7. Also, TLR4 expression increases at later embryonic days (E17 onwards). Furthermore, murine E10.5 macrophages co-express TLRs and CD14, which phagocytose apoptotic cells and bacteria, and secrete several cytokines and chemokines [36]. Hence, TLRs regulate embryonic homeostasis via iron metabolism.

The preimplantation human embryos also express TLRs [37]. Furthermore, TLR3 in the brain cells of mice in the early embryonic stages and neural stem/progenitor cells (NPCs) control the neurosphere formation. For example, NPCs from TLR3-deficient murine embryos proliferate highly and form higher numbers of neurospheres than wild-type (WT) embryos [38]. Therefore, TLR3 is a negative regulator of NPC proliferation and neurosphere formation in mice, which needs exploration in humans. TLR2 and TLR4 expression increases throughout gestation in sheep lungs, and LPS exposure increases their expression in fetal sheep lungs [39]. This study is critical as ovine and human TLR2, 3, and 4 share 83–88% homology. Human blastocysts highly express TLRs 9 and 5, and TLRs 9, 5, 2, 6, and 7 are expressed throughout embryonic development, and their stimulation in vitro produce different cytokines and chemokines such as IL-8 and monocyte-chemoattractant protein-1 (MCP-1) [37]. Therefore, it will be novel to observe the impact of TLRs in human embryonic development and TLR deficiency on reproduction or reproductive health. For example, TLRs are critical in reproductive tract inflammation, which can impair reproductive potential [40, 41]. Furthermore, human sperms express TLR2 and 4, and their activation induces apoptosis that can impair male fertility [42]. Hence, it is critical to understand TLRs’ biology in human reproduction and development.

Plenty of data are available for TLRs in a vast array of infectious (bacterial, viral, fungal, and parasitic) and inflammatory diseases [5, 13, 43, 44, 45, 46, 47, 48]. We have previously mentioned TLRs’ role in autoimmune and PIDs. Therefore, this section will introduce their role in neurodegenerative diseases (NDs) and cancers. For example, Alzheimer’s and Parkinson’s disease (AD and PD) incidence has increased worldwide since their first report. PD patients’ number has more than doubled from 1999 (2.5 million) to 2016 (6.1 million) worldwide, which is further increasing [49]. For example, in the United States, 6.1 million people may have AD as per data collected by the Alzheimer’s Disease Association (ADA) in 2022, and according to PD foundation, over a million people have PD.

Inflammation plays a significant role in ND pathogenesis so as TLRs do [12, 50, 51, 52, 53, 54, 55, 56]. A novel variant p.E317D in the TLR 9 gene, co-segregating with early-onset AD (EOAD) in an autosomal dominant manner, identified in a Flander-Belgian family, increases AD risk by compromising innate immunity-mediated protection [57]. The p.E317D TLR9 variant reduces its potential to activate NF-κB by 50%, indicating that p.E317D is a loss of function mutation. The protective role of TLR9 in AD comprises the release of anti-inflammatory cytokines and upregulation of Axl (a negative regulator of TLR-mediated pro-inflammatory signaling), Run domain Beclin-1 interacting and cysteine-rich containing protein (RUBICON, an autophagy suppressor), and associated signaling pathways regulation microglial phagocytic function and inflammatory signaling [57]. For example, microglia and myeloid cell-specific deletion of RUBICON in mouse genetic models of AD induce early onset of neurotoxic amyloid-beta (Aβ) plaques, microgliosis, and increase in pro-inflammatory cytokines in cortex and hippocampus [58, 59]. AD patients’ brains express lower RUBICON, Atg16L, and Atg5 levels than non-AD brain samples [60]. Furthermore, TLR9 activation ameliorates vascular amyloid pathology in mice and provides cognitive benefits [61]. The increased TLR4 expression and its co-localization with pSer129 αSyn and Iba-1in glial cells of substantia nigra (SN) and medial temporal gyrus (GTM) in PD patients can be targeted to prevent inflammatory neuronal damage [62, 63].

Autophagy is critical for Aβ secretion and plaque formation, and TLRs are known to control autophagy [64, 65, 66]. For example, TLR7-mediated autophagy increases epileptic susceptibility by reducing kinesin family member 5 A (KIF5A)-dependent gamma-aminobutyric acid (GABA)A receptor transport in mice [67]. Furthermore, TLRs are critical players in non-NDs of the brain called neurological diseases due to their role in central nervous system (CNS) homeostasis and neurogenesis, including neuronal pruning, learning, and memory [68]. The recognition of extracellular Tau protein (one of the causal factors for PD and associated dementia) stimulates microglia to phagocytose live neurons via the TLR4/NLRP3/Caspase 1 axis and NADPH oxidase activation [69, 70]. The burden of neurological diseases is also increasing world-wide [71]. For example, neurological diseases ranked third after cardiovascular diseases and cancers in Europe’s total death [71]. Therefore, exploring TLRs’ role in NDs and other neurological diseases has the potential for designing novel therapeutics. For example, targeting TLRs may serve a promising therapeutic strategy against AD and PD [62, 63, 69, 72, 73, 74]. Furthermore, Clostridium butyricum improves the cognitive decline in mice with intraventricular injection of streptozotocin (ICV-STZ)-induced AD by suppressing TLR4 signaling through gut-brain axis [74]. However, C. butyricum use in humans may cause serious adverse events [75].

Cancer is another critical human health issue. The immune system and inflammation play significant roles in cancer pathogenesis, and TLRs are relevant regulators of inflammation and immunity [5, 76, 77, 78]. Thus, chronic inflammation due to prolonged TLR signaling activation may predispose to cancer development later in life. However, TLR signaling in cancer cells may exert tumor-suppressive or promoting effects, depending on the type of TLR signaling [79]. For example, TLR 2, 4, and 7/8 activation in tumor cells promotes tumor progression via creating and supporting immunosuppressive tumor microenvironment (TME) or tumor immune microenvironment (TIME), supporting resistance to apoptosis among cancer cells and their proliferation and metastasis. On the other hand, TLR2, 3, 4, 5, 7/8, and 9 activation on cancer cells in conjunction with chemotherapy and immunotherapy exerts antitumor action [79]. For example, TLR5 agonists enhance antitumor immunity and increase the efficacy of immune checkpoint inhibitors (ICI) by overcoming their resistance [80]. Furthermore, TLR activation on cytotoxic T and natural killer (NK) cells and antigen-presenting cells (APCs, macrophages, and DCs) has anticancer action [79]. Therefore, TLRs are critical in cancer immunotherapy depending on the cancer and targeted cell type [81, 82, 83]. Hence, TLRs are expanding their boundaries to cancers and neurological diseases, including epilepsy and NDs.

4. Conclusion

The story of TLRs began in 1997 with the discovery of TLR4 in human spleen, immune, epithelial, and endothelial cells as a homolog of Drosophila Toll protein that filled the long-standing gap of the microbial recognition, their phagocytosis and clearance by immune cells. After almost 30 years of TLR4 discovery and revolutionizing immunology research, TLRs are still at the top as critical PRRs and immune regulators. However, in the last 30 years, they have expanded their territory from infection, immunity, and inflammation to neurosciences, mammalian reproductive biology, and cancer. Even TLRs and DAMPs are critical in heart transplant rejection [84]. TLR agonists and antagonists have a wide range of applications in different inflammatory and infectious diseases and cancers (as adjuvants). TLR-based emerging therapeutics and vaccine candidates are under clinical trials for several diseases [85, 86, 87]. Furthermore, harnessing innate immunity for cancer and ND therapy is a critical research area, and TLRs are relevant components of innate immunity [4, 88, 89]. Hence, TLRs control innate immunity as crucial members of the PRR family with diverse immune and non-immune functions.