Tuberculosis (TB) is caused by Mycobacterium tuberculosis (M. tb), and it remains one of the major bacterial infections worldwide. Innate immunity is an important arm of antimycobacterial host defence mechanism that senses various pathogen-associated molecular patterns (PAMP) of microbes by a variety of pattern recognition receptors (PRRs). As per the recent discovery, Toll-like receptors (TLRs) play a crucial role in the recognition of M. tb, this immune activation occurs only in the presence of functional TLRs. Variants of TLRs may influence their expression, function and alters the recognition or signalling mechanism, which leads to the disease susceptibility. Hence, the identification of mutations in these receptors could be used as a marker to screen the individuals who are at risk. In this review, we discuss TLR SNPs and their signalling mechanism to understand the susceptibility to TB for better therapeutic approaches.
Tuberculosis (TB) remains an important determinant of morbidity and mortality worldwide. Mycobacterium tuberculosis (M. tb) is the causative agent of TB. The majority of infected persons remain asymptomatically (latently) infected with the pathogen, while 10% progress to active TB  due to complex environmental, genetic, and immunological interactions that are incompletely defined.
Innate immunity in tuberculosis infection
Inhalation of M. tb bacilli activates innate immune responses from pulmonary alveolar macrophages and dendritic cells (DCs) that contribute to host immunity. In the early phase of infection, M. tb, internalized by phagocytic immune cells, replicates intracellularly, and the bacteria-loaded immune cells could efficiently cross the alveolar barrier and transmits to various other extrapulmonary sites [2, 3]. The intracellular replication and simultaneous dissemination of the pathogen occur prior to the development of the adaptive immune responses. This shows the unique feature of M. tb to establish a protected niche where they can avoid elimination by the immune system and persist for ever [4, 5]. The innate immune system has various pathogen recognition receptors (PRRs) that are expressed on the cell surface, in intracellular compartments, or secreted into the blood stream and tissue fluids , which specifically recognizes the pathogen-associated molecular patterns (PAMP) for initiating and coordinating the host innate immune response . As per the recent research on PRRs like Toll-like receptors (TLRs), nucleotide oligomerization domain (NOD)-like receptors (NLRs) and other C-type lectin receptors plays an important role in the recognition of M. tb. Here, we have summarized the information available on host innate immune response especially TLRs, host–pathogen interaction and the importance of signal transduction mechanisms involved in the pathogenesis of TB.
Discovery of Toll-like receptors
TLRs are phylogenetically conserved mediators of innate immunity, which are essential for microbial recognition on macrophages and DCs [8-10]. Toll was first identified in Drosophila as a type I transmembrane receptor, which controls dorsal–ventral polarity during embryogenesis . After the identification of Toll as an essential receptor in the innate immune recognition in Drosophila, a homology search of databases lead to the discovery of a homologue of Toll in humans . It is now designated as TLR4 and is involved in the gene expression of inflammatory cytokines and costimulatory molecules . Later studies identified several proteins that are structurally related to TLR4. Currently, 11 mammalian TLRs were identified of which TLR1-10 are functional in humans.
TLRs are transmembrane proteins containing lucine-rich repeats (LRR) in their extracellular domains. The cytoplasmic domain of TLR is homologous to the signalling domain of Interleukin-1 receptor (IL-1R) known as Toll/IL-1 (TIR) domain that links to IL-1R-associated kinase (IRAK), a serine kinase that activates transcription factors like nuclear transcription factor (NF)-κB, which leads to the production of cytokines. Activation of TLR by its specific ligand may result in several possible biological outcomes, ranging from the cytokine secretion, modulation of the adaptive immune response, rapid cellular differentiation, apoptosis and direct antimicrobial activity [12-14].
Recognition of Mycobacterium tuberculosis by Toll-like receptors
Of 10 TLRs, TLR1, TLR2, TLR4, TLR6, TLR8 and TLR9 are thought to be involved in the recognition of mycobacteria. Most important M. tb cell-surface ligands that interact with TLRs and other receptors are 19- and 27-kDa lipoproteins, 38-kDa glycolipoprotein, the lipomannan (LM) and mannose-capped lipoarabinomannan (ManLAM) [15-17]. This interaction ultimately results in the activation of NF-κB and production of proinflammatory cytokines, chemokines and nitric oxide through either myeloid differentiation primary response protein 88 (MyD88)-dependant or MyD88-independent pathway [18-21]. The MyD88-dependent pathway involves the early-phase activation of NF-κB, all the TLRs except TLR3 have shown to activate this pathway. TLR3 and TLR4 act via MyD88-independent pathway with delayed kinetics of NFκB activation .
MyD88 plays an important role during myeloid cell differentiation and found to be essential for M. tb-induced macrophage activation . Ligand binding leads to TLR dimerization and conformational change, which then associates with the adaptor MyD88 and interacts with the IRAK-4 via their respective death domains [23-26]. Once IRAK-4 binds to MyD88, it recruits and phosphorylates IRAK-1, which activates the kinase function of it. IRAK-1 then autophosphorylates itself, recruiting tumour necrosis factor receptor–associated factor-6 (TRAF6) to the MyD88/IRAK-4/IRAK-1 complex. Next, IRAK-1 and TRAF6 dissociate from the receptor complex and interact with additional molecules, resulting in c-Jun N-terminal kinase (JNK) and inhibitor of κB kinase (IKK) activation. These proteins then induce activator protein-1 (AP-1) and NF-κB (P50, P65) activation, ultimately leading to the transcription of genes encoding proinflammatory cytokines such as TNFα, IL-6, IL-8, IL-1β and chemokines (Fig 1).
TIR domain-containing adapter protein inducing IFN-β (TRIF, also known as TICAM1) was found to mediate the MyD88-independent pathway. The TRIF-related adapter molecule (TRAM, also known as TICAM2) specifically acts to bridge TLR4 with TRIF [28, 29]. TLR4 and TRAM get delivered to the endosome and subsequent recruitment of TRIF precedes the initiation , which involves the non-canonical IкB kinases (IKKs), TANK binding kinase 1 (TBK-1) and IKKε/IKKi that induces interferon regulatory-3 (IRF-3) phosphorylation thus leading to the activation of IRF-3, and thereby induces IFN-β. It, in turn, activates Stat1, leading to the induction of several IFN-inducible genes [31-33]. IRF-3 may also associate with canonical IKKs composed of IKKα and IKKβ, both of which phosphorylate Ser32 and Ser36 of IкBα, thereby inducing NF-кB activation  (Fig 1).
Single nucleotide polymorphisms (SNPs)
SNPs are single-allele mutations in the genomic sequence of an organism, which are responsible for about 90% of all human DNA variation and play an important role in human evolution, drug sensitivity and disease susceptibility  Synonymous SNPs are those with different alleles encoding for the same amino acid (silent mutation). Non-synonymous SNPs (nSNPs) have different alleles that encode different amino acids. Both synonymous and non-synonymous SNPs influence promoter activity and pre-mRNA conformation (or stability). They also alter the ability of a protein to bind its substrate or inhibitors  and change the subcellular localization of proteins (nSNPs).
TLR-1 is a type I transmembrane protein composed of 786 amino acids with a 581-amino acid leucine-rich extracellular domain, a 23-amino acid transmembrane domain (amino acids 582 to 604) and a 181-amino acid cytoplasmic signalling domain. It is also designated as cluster of differentiation 281 (CD281). It is expressed at higher levels in the spleen and peripheral blood cells . Human TLR1 plays an important role in host defence against M. tb. A study in Seattle and Vietnam population identified seventeen polymorphisms in the coding region, in which seven variants were synonymous C114T (H38H), A914T (H305L), C944T (P315L), T1583C (C528C), G1677A (P559P), T1760G (V587G), T1892G (L631R), and ten were non-synonymous G1968A (L656L), C2198T (P733L), T130C (S44P), A1482G (V494V), C1938T (H646H), G239C (R80T), C352T (H118Y), A743G (N248S), A1518G (S506S) and T1805G (I602S),with seven of them in the extracellular domain and two in the intracellular domain .
TLR1/2 and TLR2/6 receptor pairs exhibit different specificities towards many microbial agonists [38-40], which is determined by the region composed of LRR motifs. Recently, a study reported that there are three nSNPs located in the LRR region of TLR1. P315L is one of the nSNPs that may have impact on the innate immune response and clinical susceptibility to many infectious diseases . Studies have shown that TLR1 polymorphisms were associated with impaired cell-surface expression . R80T, N248S and I602S nSNPs were associated with invasive aspergillosis  and with Crohn's disease . In malaria and H. pylori-induced gastric diseases, 602S was found to be a risk factor [45, 46]. A study reported in Germany found that the 743A and 1805G correlate with TLR1 deficiency and impaired functionality and were strongly associated with susceptibility to TB ; similarly, in African American and European American patients, common variants like N248S and S602I and rare variants like H305L and P315L were associated with altered immune response to M.tb ligands and susceptibility to Leprosy .
In response to stimulation with the TLR1 ligand PAM3, the variants containing 602I were fully capable of mediating NF-kB signalling, while variants with SNP 602S had impaired signalling, this implies that 602I regulates lipopeptide responses. N248 (common in European Americans) is a conserved amino acid site in the extracellular domain of TLR1 and is a putative glycosylation site. Replacement of the Asn residue with Ser might result in altered glycosylation, potentially changing TLR1 folding or function  (Table 1).
Table 1. Toll-like receptor polymorphisms and their effect on host immune response
TLR2 is encoded by a DNA sequence composed of 2352 bases that codes for 784 amino acids . This type I transmembrane receptor is characterized by an extracellular leucine-rich domain (amino acids 1–588), a single transmembrane domain (amino acids 589–609) and a cytoplasmic domain (amino acids 610–784). It is found to be the principal mediator of macrophage activation in response to M. tb . It recognizes 19-kDa lipoprotein that leads to the production of inflammatory cytokines, such as tumour necrosis factor-α (TNF-α) and interferon (IFN)-γ, that are predominantly secreted by T-helper-1(Th1) cells [49-52]. From several studies, it has become clear that phagocytosis does not lead to immune activation in the absence of functional TLRs. So, polymorphism in the TLR2 gene may lead to decreased response of macrophages to bacterial peptides, resulting in an attenuated immune response in the host . It has been reported that 89 SNPs were identified in TLR2 gene. Many SNPs have no effect on cell function, and there is limited literature available on functional polymorphisms of TLR-2. Among 17 functional polymorphisms described till now, nine of them are nSNPs . The extracellular domain of TLR2 is crucial, which specifically binds to various ligands, and for dimerization with TLR1 or TLR6 [55, 56], five SNPs were identified in that region [57-59], three of them (Arg753Gln, Arg677Trp and Pro681His) have been linked to reduced NF-kB activation and to increased risk of infection, by blocking TLR2 binding with MyD88 [24, 54]. Pro681His is present in Asian and African populations, seems to be absent among white population. This missense variant has been associated with lepromatous leprosy in a Korean population  and with susceptibility to TB in a Tunisian population .
The TLR2 Arg677Trp (R677W) polymorphism inhibits both Mycobacterium leprae-mediated and M. tb-mediated NF-kB activation and production [62, 63]. A study reported that the patients carrying the TLR2 677W allele had lower basal and Mycobacterium-stimulated serum IL-12 levels, which is necessary for the activation of the IFN-γ pathway and the induction of the Th1 responses, which are vital for cell-mediated immunity. Studies have shown that prolonged TLR2 signalling by lipoproteins of M. tb inhibits major histocompatibility complex (MHC)-II expression and processing of antigens by macrophages [64, 65]. Thus, a subset of infected macrophages may be unable to present M. tb antigens to CD4+ T cells resulting in insufficient activation of effector T cells leading to evasion of immune surveillance and creation of niches where M. tb survives and persists [15, 18] (Table 1).
TLR4 composed of 839 amino acids, is activated by bacterial lipopolysaccharide (LPS) and lipotechoic Acid (LTA). Both LPS and LTA first bind to the cluster of differentiation-14 (CD14) receptor, which then transfer them to TLR4. It homodimerizes and forms a complex with the protein MD2, and this complex is then transported to the cell surface [66, 67]. The transmembrane domain (amino acids 632-839) of TLR4 has a critical role in the functional oligomerization of it, a missense mutation, Val651Phe, found in that region may alter the function of TLR4 in response to LPS . A study identified five low-frequency missense mutations (Ser73Arg, Ala97Val, Tyr98Cys, Thr175Ala and Thr399Ile) in the ectoplasmic LRR domain , which are common variants in the Caucasian population . The amino acid substitutions may alter protein structure and function, but it is still not known whether Tyr98Cys and Thr175Ala alter the function of TLR4. Ser73Arg showed a slightly higher frequency in typhoid cases . Asp299Gly and Thr399Ile SNPs were not found in Korean, Taiwan Chinese and Japanese populations [71, 72]. Thr399Ile occurred in a low frequency in the Vietnamese population. Asp299Gly variant is associated with TB susceptibility in HIV-infected patients in Tanzania , and there is no association between TLR4 Asp299Gly and TB susceptibility in Gambian  and Mexican population .
The two cosegregated mutations Thr399Ile and Asp299Gly, which lies in the ectoplasmic LRR domain, are significantly associated with a decreased cytokine response to LPS  stimulation and increased susceptibility to a variety of infections [77-80] by affecting the extracellular domain of the TLR4 receptor . These LRR region mutations may potentially disturb phosphorylation of TLR4 altering downstream signalling of inflammatory mediator activation, ultimately contributing to disease susceptibility . Thus, individuals who have these variations in TLR4 may prone to develop TB (Table 1).
Toll-like receptor 6 consists of 796 amino acid polypeptide containing only one exon . It is expressed in the spleen and peripheral blood leucocytes and is a coreceptor for TLR2. TLR-6 activated through MYD88 and TRAF6, leading to NF-kB activation, cytokine secretion and inflammatory response. It recognizes lipid-containing ligands like lipoteichoic acid, diacylated lipopeptides like PAM2 (PAM2CSKKKK, S-[2,3-bis(palmitoyloxy)-propyl]-(R)-cysteinyl-(lysyl)3-lysine) , and it also recognizes soluble tuberculosis factor (STF), Borrelia burgdorferi outer surface protein A lipoprotein (OspA-L) and phenol-soluble modulin (PSM) with TLR2. Polymorphisms in the coding region of TLR-6 gene were investigated in Chinese Cantonese population, a total of seven SNPs were detected, five of them with amino acid substitution, (Met59Thr (+176T/C), Ile120Thr (+359T/C), Val327Met (+979G/A), Val465Ile (+1393G/A) and Val470Leu (+1408G/T). Remaining two are (+1083C/G and +1263A/G) without amino acid substitution. An nSNP, +745T/C (Ser249Pro) was significantly associated with protection from asthma in African Americans and European Americans. In African Americans, homozygote for the common variant TLR6 249S had a significantly increased risk for TB disease .
C745T and G1083C were associated with decreased IL-6 in response to lipopeptide stimulation in a whole blood cytokine assay , and 745T variant mediates less NF-κB signalling in a transfected HEK cells in response to stimulation with diacylated lipopeptide or Mtb cell lysate . A study reported that 745T and 1083C were associated with increased IFN-γ or IL-2 levels after BCG vaccination , but the mechanism is still unclear (Table 1).
TLR8 is located on X chromosome and able to recognize single-stranded RNA from pathogens such as RNA viruses. According to the literature, Davila et al.  first reported TLR8 SNPs, and they have analysed 149 SNPs from Indonesian and Russian pulmonary TB patients, of these four SNPs were significantly associated with the pulmonary TB among Indonesian and Russian males. Three of the associated TLR8 variants are −129 C/G, −2167 A/G and −1145 A/G present in the regulatory regions, and one variant 1 A/G (Met1Val) at the start codon. Indonesian males were carriers of Met1Val, allele A showed an increased susceptibility to pulmonary TB, While G allele shows protection from TB. Another study reported in Turkish children  also showed an association with susceptibility to pulmonary TB among male children, but found no associations with −129 C/G SNP for TB susceptibility in children, whereas Davila et al. found a strong allelic association with minor allele C in susceptibility to pulmonary TB in males, but the mechanism through which TLR8 recognizes M. tb and intracellular signalling remains unknown (Table 1).
TLR9 composed of 2 exons and encodes 1032 amino acids . It recognizes unmethylated CpG motifs in bacterial DNA. It was found to be essential for cellular responses to mycobacterial CpG DNA . In vitro studies showed that DCs release IL-12 in response to M. tb through TLR9 [89, 90]. A report demonstrates that TLR9-deficient mice are susceptible to Mtb infection, and mice lacking both TLR2 and TLR9 are more susceptible  to TB. Four SNPs, C-1486T, C-1237T, G+1174A and G+2848A, have been reported to show high heterozygocity among three major US ethnic groups . C-1237T, a polymorphism located within the putative promoter region that may influence transcriptional regulation of the TLR9 gene. SNP G+1174A, located in the intron of TLR9, showed a significant association with TB in Indonesian females . Promoter polymorphisms, namely −1237C/T and −1486C/T, are not associated with pulmonary TB in south Indian population .
TLR9 activation is essential for the maintenance of M. tb Ag elicited pulmonary granulomatous response; however, the underlying mechanism is not known. SNPs in promoter region potentially affect gene expression levels by altering the binding of gene transcription factors and SNPs in introns, affecting mRNA splicing and/or enhancement of gene transcription. Carvalho et al.  reported that peripheral blood mononuclear cells (PBMCs) harbouring the -1237 TC genotype shown higher expression of both TLR9 and IL-6 and increased B-cell proliferation in response to CpG DNA, but the mechanism is not known (Table 1).
Pattern recognition is an important component of the host response to infection with M. tb, which triggers inhibitory mechanisms via TLRs. The coordinated regulation of TLR signalling through their respective ligands might be important for controlling the extent of the host immune response to prevent the progression of M. tb growth. Both the extent and quality of the innate immune response are likely to be critical for control of M. tb infection. TLR polymorphisms have shown great impact on susceptibility to TB. Individuals with a particular TLR genotype may have higher or lower affinity to M. tb ligands leading to differences in signal transduction. So, further studies systematically investigating the relevance of naturally occurring mutations in the TLRs, their adaptors (MyD88, TIRAP, TRIF, TRAM) and downstream molecules such as IRAKs, TRAF6 may help to understand the molecular biology of these molecules and to assess the cumulative effect of various combinations of SNPs to obtain a stronger association with disease and also to identify high-risk individuals especially in household contacts.
We thank Staff of the free chest clinic Mahavir PPM DOTS, Tuberculosis Unit (1 T.U) Bhagwan Mahavir Trust, and Department of Biotechnology, Government of India. Sanction order no: BT/01/COE/07/02, dated 30/12/08, DBT. Sanction order no: 102/IFD/SAN/3209/2012-2013, dated 28/09/12, DBT.