The innate immune system, being the first line of defense, employ a variety of transmembrane and secreted molecules, known as pattern recognition receptors (PRRs), for the activation of adaptive immunity, apoptosis, opsonization, activation of complement system and induction of pro-inflammatory mediators etc. Among the PRRs, Toll like receptors (TLRs) occupies a prominent place that was initially described as being involved in embryonic development of Drosophila . Later, when, a homologue of Drosophilla Toll, TLR4 was cloned in 1997, it was confirmed that Toll signaling pathways is conserved in human and it plays important role in adaptive immune activation . Soon after its discovery, it became evident that lipopolysaccharide (LPS), a bacterial component, is being recognized by TLR4, a PRRs, establishing a link between pathogen associated molecular patterns (PAMPs) and TLRs [3-5]. PAMPs are conserved molecular signatures found on different microbial organism like bacteria viruses, fungi and protozoa. Different PAMPs are recognized by different TLRs like LPS and lipoteichoic acid (all recognized by TLR4); peptidoglycans (cell walls), lipoproteins (bacterial capsules) and zymosan (all recognized by TLR2 following heterodimerization with TLR1 or TLR6); flagellin (in bacterial flagella, recognized by TLR5), unmethylated bacterial or viral CpG DNA (recognized by TLR9) and viral RNA (single-stranded recognized by TLR7 and TLR8, double stranded by TLR3).
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Toll like receptors are classified as the members of Toll/IL-1 receptor superfamily (TIR) based on their structure similarities; they all share an ectodomain composed of leucine-rich repeats for recognition of PAMPs, an intracytoplasmic TIR domain that participate in the recruitment of adaptor molecules such as MyD88, MAL, TRIF, TRAM etc, and initiation of downstream signaling and a transmembrane domain. their expression pattern is highly selective based on their PAMPs recognition like TLR 1, 2, 4, 5 ,6 are predominantly expressed on cell surface as they involved in recognition of cell surface molecules, while TLR 3, 7 and 9 expressed in endosomal compartments because they are involved in nucleic acid recognition [6, 7]. Some cell-surface TLRs are internalized after ligand binding; TLR2, for example, is recruited to macrophage phagosomes .
Upon engagement of TLRs with their cognate ligand, TIR domain recruits adaptor molecules and induces interferon (IFN) and pro-inflammatory cytokines such as TNF-α, IL-1, IL-6 . The expression of different set of cytokines depends on the cell type being activated. Stimulation of human TLR7, for instance, induces IFN-α from plasmacytoid dendritic cells (pDCs) important for innate antiviral immunity and the development of adaptive immunity, whereas it induces IL-12 from myeloid dendritic cells (mDCs), associated with the induction of a Th1 response . Their differential induction of cytokines can influence the therapeutic advantages of many TLRs agonists as observed, at least in case of murine TLR4 [10, 11]. In spite of variable cytokine expression in different DC subset, TLR agonist and signaling adaptors, TLR activation generally results in activation and phenotypic maturation of all DCs.
TLRs are evolutionary conversed receptors mostly expressed on the cell surface of innate immune system providing them organism-wide sensing system; because of this their expression pattern is devoted according to the functional role of each cell. These differential expressions confer variation, when comparing with other species, in their expression and regulatory mechanism, for instance mice , placing important limitations on the interpretation of animal model studies.
Up till now, 10 different TLRs have been reported in humans that express on different immune and non-immune cells. mDCs express TLR1-6 and 8 whereas pDCs express TLR7 and 9 only [9, 12-15]. TLR7 expression has not been fully understood with reports on both mDCs and monocyte-derived DCs (moDCs) [12,14]. Neutrophils and monocytes express all TLRs except TLR3 while TLR1 is the only receptors expressed by natural killer (NK) cells, and B lymphocytes express TLR9 and 10 [16-19]. The expression pattern in T-cells is a little bit different, as activated T-cell subsets; including memory cells, express TLR2 as a costimulatory receptor , whereas regulatory T cells (Tregs) can express TLR8 and 10 [21, 22]. While TLRs are primarily expressed in hematopoetic cells, they have also been described in keratinocytes  and epithelial cells of the intestinal, urogenital and respiratory tracts [24-26], and are likely to provide antimicrobial defense in addition to the mechanical barrier of the epithelial layer.
TLRs not only play role in microbial identification, but also immune sensing against the self-altered cancer cells. A recent study has reported a link between polymorphism in TLR4 gene not only linked with microbial sensitivity but also with the susceptibility of cancers. For instance, the individuals with TLR4 T399I mutation have a higher risk to develop nasopharyngeal carcinoma (NPC) . Also, the association between TLR2 and MZL is conceivable given the strong evidence linking Helicobacter pylori to the pathogenesis of mucosa-associated lymphoid tissue lymphoma, a common subtype of MZL, and the role of TLR2-modulated immune responses to this pathogen [28, 29].
Physiological Importance of Ð¢LRs
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Physiologically, TLRs are critical in induction of innate and adaptive immune system against the invading microorganisms after detecting the PAMPs [30, 31]. These receptors mount immune response against pathogens in both skin and mucosa of respiratory, gastrointestinal and urogenital tracts. They activate multiple pathways leading to the secretion of anti-inflammatory molecules (including TNF-α, IL-1, IL-6 etc.) and chemokines (MCP-1, MCP-3, GM-CSF, etc.). TLRs have been correlated with the transcriptional and posttranslational regulation (proteolytic cleavage and secretion) of antimicrobial factors, such as defensins α and β, phospholipase A2, lysozyme, and so on . Also, they are being involved in opsonization of microorganisms and optimize their inactivation by regulating the release of peroxy radicals and nitric oxide [33, 34]. It is now known that TLRs on the endothelial cells surface indirectly promote the leukocyte homing into the inflammatory region by enhancing the expression of leucocyte adhesion molecules E-selectin and ICAM-1 .
Different antiviral molecules such as INF-α and β are also overexpressed by TLRs in hematopoietic and stromal cells to combat the antiviral infection . Moreover, as described by recent reports, TLRs lead to apoptosis via the production of certain pro-apoptotic factors (FADD, caspase-8, protein kinase R) which accounts for an important defense mechanism against certain incurable pathogenic insults . TLR-dependent activation of professional antigen-presenting dendritic cells is determinative in several essential processes providing the development of the adaptive immune response, such as the activation of mature T-cells, processing and presentation of microbial antigens, elevation of the expression of the costimulatory molecules (Ð¡D80, CD86) required for the activation of naive CD4+-T-cells, and suppression of regulatory T-cells via IL-6 production 24. The TLR-dependent activation is also important for B-cell proliferation and maturation during the infection process .
Thus, these accumulating evidences suggest an important role in initiating and development of the inflammatory process (activation of innate immune reactions) in response to the introduction of various pathogens (including protozoa, fungi, bacteria, and viruses). Furthermore, it is now believed that pathogen recognition by TLRs is the midpoint in the initiation of the adaptive immune response, which is the second line of defense . Apart from their protective role, TLRs also implicated in the pathogenesis of autoimmune diseases (systemic lupus erythematosus), arthritis, atherosclerosis, and certain other disorders . Recent data poin to the dual function of these TLRs as either activate antitumor immunity [41, 42] or, on the contrary, favor tumor progression [43, 44]. During the past few years, different studies delineate the role of TLRs in cancer elimination or progression, but still further studies are required to precisely define their functional aspects. Their role might be cell and its context dependent where it is going to express. Another aspect is the influence of extracellular matrix, other co-receptor being affected by TLR signaling and the cross talk among different cell signaling, that might modulate the TLR signaling (Reference).
Regulation of TLRs
After being activated and establishing a pro-inflammatory response against the microbes and other potential harmful molecules, there must be some point when the signaling through TLRs abolish and the system should return to its normal physiology to avoid a potential deteriorating response towards the self-antigens. The regulation of TLR signaling takes place at multiple levels as well as in various ways. The well-established phenomenon of feedback inhibition frequently observed in the various signaling pathways and in TLR signaling as well. When TLRs get activated, it stimulates the inflammatory cytokines and some other molecules that negatively inhibit its own signaling resulting in restoration of homeostatic condition. In the following section, we will try to highlight some important mechanisms that are involved in negative regulation of TLRs to maintain a balanced condition in the cellular system.
To date, many negative regulators have been identified and characterized , and we have tried to comprehensively address few ways of negative TLR regulation. These have been included from receptor regulation, adaptor complex destability, ubiquitination and phosphorylation and transcriptom control.
1) Soluble and decoy factors in TLR regulation
During the bacterial challenge, if the innate and adaptive immunity do not respond properly, it may lead to septic shock and death of the patient. As part of activation, the sustained and over-activation of TLRs also result in abnormality and autoimmune condition. Therefore, it is mandatory to balance the pro- and anti-inflammatory responses. Soluble receptor is a good way to modulate the immune response. In a study, it has been reported that monocytes constitutively maintain the intracellular TLR2 level and secretes soluble TLR2 (sTLR2) that hinders the ligand interaction with TLR2. Cells secrete higher level of sLTR2 when they get activated . The soluble form is a natural and resulted from a post translation modification inside the cell.
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The soluble form of TLR4 (sTLR4) has also been reported to inhibit the TLR4 mediated signaling, possibly by interfering with receptor ligand association . The study was carried out in mouse originated macrophage cell line that the presence of sTLR4 significantly reduced the activation of NF-kB leading to the conclusion that sTLR4 might involve in regulating the TLR signaling.
Soluble CD14 (sCD14) and sST2 are also involved in regulating the TLR4 and TLR1, 4 induced cytokine release respectively [47, 48]. The sCD14 directly binds to LPS and alter the signaling pattern while sST2 inhibits the overexpression of TLR1 and 4. The hypo-expression leads to the lower production of proinflammatory mediators.
Apart from soluble factors, there are certain other receptors that may also involve in negative regulation of TLR signaling such as SIGIRR, ST2L and RP105.
Single immunoglobulin IL-1R-related molecule (SIGRR) belongs to Toll-like receptor-interleukin 1 receptor signaling (TLR-IL-1R) receptor superfamily and is important in negatively regulating the signaling. This protein binds to TLR-IL-1R components in a ligand dependent manner and hinders the recruitment of downstream signaling molecules . Mouse deficient of SIGRR, when stimulated with ligand produced higher volumes of inflammatory cytokines indicating the regulatory importance of SIGRR.
The membrane bound form of ST2L is also keeps a check on the over-activation of TLR4 and TLR2, 4 and 9 by preventing the downstream adaptor complex through its BB loop [50, 51]. Experiments with mice lacking ST2L fail to develop the immune tolerance towards the ligands resulting in fatal consequences.
RP105 is the homologue of TLR, lacking the signaling domain, specifically inhibits the signaling through TLR4. RP105 along with its helper molecule MD-1 bind to TLRs ectodomain and prevent ligand binding to TLR4 receptor and inhibit signaling. It is expressed on different antigen presenting cells. Cells deficient of RP105 showed a high response to LPS implying a negative role of this protein .
2) Negative regulators of adaptor complex
Dissociation of adaptor molecules at TIR-domain complex is a profound mechanism to control the inflammatory response when ligands bind to receptors. Several variants of adaptor molecules have been identified as the negative regulator of the complex formation and prevent downstream signaling pathways. Among them, TRAM adaptor with GOLD domain (TAG) is the variant of TRAM that competes with TRAM for TRIF binding. Once in bound form, it inhibits the TRIF dependent pathway . Also, to prevent the over-activation of TLR4 as a result of LPS induction, TAG is involved in internalization of TLR4 to the endosomes for degradation. In association with TMED-7 (a GOLD domain-containing protein), TAG inhibits TLR4 signaling from the endosome upon LPS stimulation .
Several proteins are involved in efficient signaling of TLRs such as MyD88, TIRAP, TRIF and TRAM , providing an opportunity to have a fine control mechanism. Another TIR domain containing protein, sterile alpha- and armadillo-motif-containing protein (SARM), also involved in blocking of TRIF complex formation by directly binding and making TRIF unavailable after LPS treatment . However, sterile α-motifs (SAM) domain is necessary for this inhibitory function, but the molecular mechanism of how this domain suppresses the TRIF-dependent pathway is largely unknown. After interacting with MyD88, IRF5 induces a set of TLR-inducible genes , but similarly IRF4, induced by TLR activation, competitively binds to MyD88 that result in inhibition of IRF5-dependent gene induction . Mice lacking IRF4 protein showed increased sensitivity to DNA-induced shock along with increased cytokine production.
Caspase-8, an important player in apoptosis, also plays a prominent role in immune cell activation using various receptors including TLRs [59, 60]. Caspase-8 activity is being modulated by tumor necrosis factor, alpha-induced protein 8 (TNFAIP8), TNFAIP8-like 2 (TNFAIP8L2; also known as TIPE2), which in turn regulates the activator protein-1 (AP-1) and NF-kB activation . TIPE-2 deficient mice and cell lines are hypersensitive to TLR activation, which is overcome by inhibiting caspase-8. This observation suggested that TIPE-2 is also involved in TLR signaling through modulation of caspase-8 .
Nucleotide binding oligomerization domain (NOD)-like receptor (NLR) family member X1 (NLRX1) was previously identified as an inhibitor of IPS-1, an adaptor for RIG-I-like receptors (RLRs), which can suppress TLR signaling through interactions with TRAF6 and IKK complex . NLRX1 undergoes K63-linked polyubiquitination after LPS treatment and dissociates from TRAF6, resulting in binding to the activated kinase domain of IKKb via the leucine-rich repeat (LRR) domain. NLRX1-knockdown mice show hyper-activation of NF-kB, elevated production of inflammatory cytokines, and increased susceptibility to LPS-induced septic shock, suggesting that NLRX1 functions as a negative regulator of TLR signaling in vivo. However, there is some disagreement concerning in vivo functions of NLRX1, at least in RLR signaling . In contrast, another NLR protein, NLRC5, negatively regulates TLR and RLR signaling through IKK inhibition . However, NLRC5-deficient mice develop normal responses against bacterium and virus infection . Further studies are required to evaluate the biological significance of these proteins.
Role of Ubiquitination/deubiquitination in signaling attenuation
Ubiquitination is a principal way to shut down signaling in cell at different stages. Ubiquitin protein marks different other proteins for degradation using ubiquitin ligase, while de-ubiquitination leads to the stability of substrates. This phenomenon is important in cell signaling regulation. It has been reported that TANK binds to TRAF-binding protein and activate both NF-kB and IRFs in vitro. However, TANK-deficient mice have shown increased NF-kB activity in response to TLR stimulation and rapidly proceed to fatal glomerulonephritis . TRAF6 ubiquitination, which is required for NF-kB activation, is enhanced in TANK-deficient cells, indicating that TANK binds to TRAF6 and inhibits its ubiquitination.
A recent study reported that small heterodimer partner (SHP, also known as NR0B2) has been implicated as a negative regulator of TLR signaling by preventing TRAF6 ubiquitination . Cells lacking SHP protein secrete higher level of tumor necrosis factor (TNF), IL-1b and IL-6 following LPS treatment. Moreover, when SHP deficient cells were injected bone marrow derive cells that express SHP elevated the protection level in mice from LPS induced lethal shock. A negative feedback loop was observed as TLR stimulation induces SHP expression through AMP-activated protein kinase (AMPK) activation- dependent intracellular Ca2+ influx which ultimately regulates the TLRs signaling .
In the cell signaling process, after the signal has conveyed, the inducer protein should be eliminated/inhibited to prevent the exaggeration in response towards that stimuli. Ubiquitin mediated degradation of several proteins is the conspicuous mechanism in signal transduction biology and in immune responses . This class comprised of several proteins among them suppressor of cytokine signaling (SOCS) of E3 ubiquitin ligase family is most widely studied that facilitate degradation of TIRAP/MyD88-adaptor-like (MAL) or TRAF proteins . By knocking out Tyro3, Axl and Mer (TAM) receptor tyrosine kinase from the DCs, it was observed that TLR stimulation produced higher level of inflammatory cytokines as compared to wild type cells. Also, activated TAM receptor in DCs down-regulate the TLR-induced cytokine production. Further studies have shown that in response to TAM receptor signaling, SOCS1 and SOCS3 get overexpress through signal transducer and activator of transcription (STAT) 1, providing a justification of TAM receptor-mediated inhibition of TLR-induced cytokine production.
In another study, it was shown that the protein Mal, which is specifically involved in signaling via TLR2 and TLR4, becomes phosphorylated by Bruton's tyrosine kinase (Btk) leading to the interaction with SOCS-1, which promotes polyubiquitination and subsequent degradation of Mal. Removal of SOCS-1 regulation potentiates Mal-dependent p65 phosphorylation and transactivation of NF-kB, leading to amplified inflammatory responses. This study implies a distinct regulation of TLR signaling . The activation of Mal for a short time reflects a potential control over the innate immune system.
An ovarian tumor domain containing deubiquitin protein, DUBA (deubiquitinating enzyme A), has also been reported as the suppressor of type 1 IFN production . DUBA selectively mediates the cleavage of K63-linked polyubiquitin chains from TRAF3 to suppress TLR-induced type I IFN production rendering NF-kB activation unaffected.
PDZ and LIM domain protein 2 (PDLIM2), a Janus kinase (JAK)/STAT inhibitor, also hinders TLR signaling by degradation of the NF-kB component p65 . This inhibitory effect is mediated by promoting K48-linked polyubiquitination on p65 and isolating it to nuclear promyelocytic leukemia (PML) bodies abundant with 26S proteasome. After stimulation of TLR, PDLIM2-deficient cells fail to degrade p65, culminating in high level of inflammation.
IRF3 and IRF7 have been reported to be controlled by ubiquitination mediated by peptidyl-prolyl isomerase Pin1. Pin1 binds to phosphorylated IRF3 and inhibit the type 1 IFN and antiviral response . It has been hypothesized that binding and phosphorylation brings conformational changes in the IRF3 structure leading to ubiquitination degradation of the protein rather Pin1 acts as an E3 ligase. As opposing to this, Pin1 found to be a positive regulator in plasmacytoid DCs where it activates the IRAK1 during TLR7 and TLR9 signaling leading to the production of type1 IFN .
A recent study reported that homologous to E6-AP carboxyl terminus (HECT) type replication and transcription activator (RTA)-associated ubiquitin ligase (RAUL) has been linked to catalyze directly ubiquitination of IRF3/7 and negatively affect type I IFN responses . Kaposi's sarcoma-associated herpes virus (KSHV) RTA promotes deubiquitination of RAUL self-ubiquitination by recruiting USP7 (also known as herpes virus-associated ubiquitin-specific protease, HAUSP), leading to the stable function of RAUL and effective degradation of IRFs to mute antiviral responses.
Further going into the deep of cell signaling regulation, it has been found that A20 also negatively affect the TLR signaling. Study indicated that the mice with A-20 deficiency display inflammation in various organs in the body and double knockout mice with A-20 and MyD88 deficiency do not manifest any such symptoms. The administration of antibiotic suppresses cachexia caused by the loss of A20 indicating that A20 might significantly suppress inflammation caused by the intestinal bacteria . Also, it helps in removal of K63 linked polyubiquitin chain to TRAF6 and aids mice to survive the LPS-induced endotoxin shock. Moreover, A20 has been shown to inhibit IKK activation by TAK1 without DUB activity, suggesting that A20 regulates NF-kB activation via multiple mechanisms .
Phosphorylation of signaling proteins in negative regulation
TLR4 signaling can also be suppressed by mitogen and stress activated protein kinase (MSK) 1 and 2 activated in the mitogen-activated protein kinase (MAPK) cascade . In cellular environment devoid of MSK1 and MSK2 hinders the binding of phosphorylated transcription factors cAMP responsive element binding protein 1 (CREB) and activating transcription factor 1 (ATF1) to their cognate promoters. The binding of these proteins to promoters activate the anti-inflammatory cytokine IL-10, and MAPK phosphatase dual specificity phosphatase 1 (DUSP1) that promotes p38 deactivation after LPS stimulation. Knockout mice for MSK1 and 2 are undergo hyper-responsive state upon LPS treatment resulting in endotoxin shock and show inflammation for a longer period of time in a model of toxic contact eczema induced by phorbol 12-myristate 13-acetate .
Several signaling pathways require the activation of MAP kinases and NF-kB that is being carried out by TGF-b-activated kinase 1 (TAK1). Earlier, only positive role of TAK1 for NF-kB and MAP Kinases has been known in lymphocytes and hematopoietic cells, while the specific other functions of TAK1 in myeloid cells have only recently been elucidated . As oppose to its positive role, TAK1 is being held responsible for phosphorylation of p38 and production of cytokines and reactive oxygen species (ROS) induced by LPS. Myeloid lineage-specific TAK1-deficient mice show splenomegaly and lymphadenopathy, and are susceptible to LPS-induced septic shock. These phenotypes are rescued by specific ablation of p38, suggesting that TAK1 negatively controls p38 activation.
To regulate the TLR signaling, cells have developed many mechanism such as to phosphorylate the target protein or to mark it for degradation using ubiquitination. Scr homology 2 domain containing protein tyrosine phosphatase 1 and 2 (SHP-1, -2) are considered to be involved in regulation of pro-inflammatory signaling as SHP-1 deficient mice rapidly develop inflammation induced lesions in a Myd-88 dependent pathway . IRAK1 and IRAK2 activities have been suppressed by SHP-1 leading to switch in production form pro-inflammatory cytokines to Type 1 interferon. This observation is in consistent with the study that the IRAK-1 is indispensable for the activation of NF-kB and inhibition of type 1 IFN.
Leishmania infection promotes binding between SHP-1 and the conserved immune-receptor tyrosine-based inhibition motif (ITIM), Kinase Tyrosyl-based Inhibitory Motif (KTIM), in the kinase domain of IRAK-1 to suppress innate immune responses . In contrary to SHP-1, SHP-2 suppresses the TRIF-dependent type I IFN production . Although, SHP-2 phosphorylates the TBK-1 protein, but its phosphatase activity is dispensable for TBK1-activated gene expression. Thus, SHP-2 acts as an antagonist rather than a phosphatase for TBK1.
Taken together, competitors of adaptors, phosphatases and DUBs disrupt the formation of adaptor complexes. Interestingly, multiple regulators target TRAF6. It is still unknown whether these regulators collaborate with each other.
In the regulation of immune response, Immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptors play an important role also by interacting with other receptors such as TLRs . Calcium signaling induced by b2 integrins and Fcg receptors causes upregulation of IL-10 and signaling molecules involved in the inhibition of innate immune responses, resulting in indirect inhibition of TLR signaling . Moreover, spleen tyrosine kinase (Syk) activated by CD11b an integrin that phosphorylates MyD88 and TRIF and facilitates its ubiquitin mediated degradation through the ubiquitin-proteasome system . Interestingly, in this regulatory mechanism, TLR-induced activation of PI3K activated outside-in integrin signaling and triggered negative regulation. By contrast, it was also shown that CD11b positively control the recruitment of TIRAP to the plasma membrane via phosphatidylinositol 4,5-bisphosphate (PIP2) and activation of cell surface-localizing TLRs , demonstrating a dual role of integrin in TLR signaling.
Feedback inhibition in TLR signaling
In the regulation of TLR signaling, feedback inhibition share a significant burden. Different researchers reported various downstream proteins that, in response to TLR stimulation, get induced and regulate the TLR signaling in negative way. Different experiments involving TRIM30a proved that this protein acts as inhibitors of TLR signaling. TLR also induce the expression of TRIM30a, a member of tripartite-motif containing (TRIM) protein superfamily that enhances the degradation of TAK1 binding protein 2 (TAB2) complex with TAB3 and TAK1 . A decrease in the availability of TABs caused reduced NF-kB activation and cytokine production. In support of these observations, TRAM30a transgenic mice and in vivo knock down studies have proved the protective effects of TRAM30a in case of LPS induced endotoxin shock. Another closely related protein, TRIM30a, has an inhibitory effect on TLR signaling by promoting the ubiquitination of TRAF6 .
Besides, ubiquitin-proteasome system, there is another way to control the signaling route. This way account for autophagy that is a major degradation system and occupy a prominent position in host defense . Knockout of autophagy related protein, Atg16L1, a Crohn's disease risk allele, paved the way for the production of high levels of Reactive Oxygen Species (ROS), IL-1b and IL-18 induced by LPS . These are TRIF dependent molecules, employing that Atg16L1 suppress TRIF-dependent pathway leading to caspase-1 activation. It has been shown that Atg16L1-deficient Paneth cells show increased expression of genes involved in intestinal injury responses .
Cylindromatosis (CYLD), a tumor suppressor deubiqutination protein has been reported to modulate TLR2 signaling . As a matter of fact, TLR2 when activated also induce the expression of CYLD that mediate the removal of polyubiquitin chains from TRAF6 and TRAF7, both of which are indispensable for NF-kB and MAPK activation by TLR2 ligands. A recent study reported another protein, USP4 that negatively regulate DUB, by binding with TRAF6 and suppresses IL-1b induced NF-kB activation . Ubiquitin specific peptidase 4 (USP4) removes polyubiquitin chains on TRAF6 in a DUB activity-dependent manner. Loss of USP4 enhances cytokine production mediated by LPS and IL-1b.
As described above, degradation of signal proteins mediated by the ubiquitin-proteasome and autophagy systems plays crucial roles in negative regulation of TLR signaling. Unlike in the case of disruption of adaptor complexes, these degradations are irreversible, suggesting that this mechanism contributes largely to termination of TLR signaling.
Recent advances in molecular genetics techniques and methodological refinements have unveiled new aspects regarding transcriptional gene regulation in different cell processes and inflammatory responses . Chromatin remodeling and modifications are important ways to control gene regulation. Cyclic AMP-dependent transcription factor (ATF-3) facilitates the deacetylation of promoter region of proinflammatory genes rendering it difficult to bind with transcription factors by recruiting histone deacetylase 1 (HDAC1) .
ATF3 deficient macrophages secrete higher amounts of IL-12p40, IL-6 and TNF-α when treated with LPS. TLRs also induce the expression of certain negative regulators like IkBNS, IkB protein, inhibitor of kappa light polypeptide gene enhancer in B cells, delta (IkB delta; also known as IkBNS), that lowers the inductions of IL-6 and IL-12p40 but without affecting TNF-a transcription . The reason behind this phenomenon is that IkBNS selectively binds to IL-6 promoter but not the TNF-a promoter. Knockout mice model are more prone to develop endotoxin shock and intestinal inflammation indicating important role of IkBNS in inflammation.
Different members of IkB family performing important physiological phenomenon in cell like B-cell CLL/lymphoma 3 (Bcl-3) is important for TLR tolerance . This protein causes insensitivity in response to prolonged TLR stimulation while Bcl-3 deficient macrophages and DCs produce large amount of cytokines when comparing to normal. This observation suggests that Bcl-3 limit the pro-inflammatory response by stabilizing p50 which occupies an NF-kB DNA binding site.
Nuclear receptors are also taking part in controlling immune response, principally, by means of monitoring gene expression. An orphan receptor, nuclear receptor related 1 protein (Nurr1) form a complex with p65 when cell is triggered by LPS. This complex recruits CoREST complex and result in transcriptional repression of various genes . Microglia cells are myeloid cells in central nervous system and respond to infection and tissue injury. Nurr1-deficient microglia cells showed hyper-responsiveness to the stimulus indicating a protective role.
Aryl hydrocarbon (Ah) receptor exist in cytosol and sense the presence of chemical compounds and play role in mounting immune response . In macrophages, these receptors get stimulated by LPS, interacts with STAT1 and NF-kB in the IL-6 promoter to hinder IL-6 production . Deficiency of Ah receptors leads to LPS induced lethal shock demonstrating a negative role of these receptors in protecting cells.
Stability of RNA is also a major factor in controlling the effect of different genes. This is mediated by several RNA-binding proteins containing CCCH-type zinc finger motif. These proteins reduce the level of inflammation by degrading corresponding mRNAs. Tristetraprolin (TTP) mediates the deadenylation of TNF-α mRNA by binding to 3' untranslated region (UTR) through deadenylase recruitment, leading to disruption of mRNA . With the help of RNase activity, Zc3h12a, a TLR inducible RNase, degrades IL-6 and IL-12p40 mRNA and ameliorate the immune response towards various TLR ligands protecting the animal from autoimmunity .
Another independent study reported that mice, deficient of Zc3h12a, show exaggerated response implicating a negative function of Zc3h12a . The mechanism through which it exhibits its regulatory role is the DUB that removes ubiquitin label form TRAF. Yet, another study reported its regulatory behavior in controlling the immune response by IKKs through phosphorylation and proteasomal degradation . Altogether, these observations assign multiple roles to the Zc3h12a in controlling immune response.
MicroRNA in signaling Regulation
In recent years, non-coding RNAs gain much importance in gene regulation. Several non-coding RNAs including miRNA have been identified as induced by TLRs and precisely control the response . These miRNAs often have several targets and a bidirectional function, similar to miR155.
miR-155 is a TLR dependent miRNA  that shows dual physiology in immune responses [107-114]. miR-155 disintegrates mRNAs of MyD88, TAB2 and IKKi to suppress TLR signaling [107-111], while it targets SHP-1  and enhances signaling. MiR-146a is induced in response to LRS stimuli and downregulate expression of TRAF6 and IRAK1 mRNA . MiR-146a deficient mice manifest the symptoms of myeloid sarcomas and chronic myeloprolifieration due to regulatory unbalance of NF-kB . It has also been reported that, soon after birth, miR-146a was express at higher level in intestinal epithelial cells to protect the gut mucosa from bacterium induced damage .
Programmed cell death protein (PDCD) 4, an apoptotic stimulus-induced tumor suppressor, has proinflammatory functions by regulating NF-kB activity and IL-10 production. It has been demonstrated that PDCD4 is crucial for regulation of responses to LPS . miR-21 is induced by LPS stimulation and negatively regulates inflammatory responses by decreasing PDCD4 expression levels. Unlike other mechanisms for TLR inhibition, transcriptional regulation often enables control of a particular subset of TLR target genes without termination of TLR signaling. This property possibly contributes to regulate the balance of immunity beyond suppression of TLR signaling.
Evasion by pathogens
Pathogens, including viruses and bacteria, have acquired certain potential strategies to escape form immune response . Hepatitis C virus (HCV) degrades TRIF to suppress TLR signaling through NS3-4A protein that has serine protease activity . Vaccinia virus (VACV) proteins A46R and A52R hinder the complex formation at TIR domain and signaling through TRAF6 and IRAK2 respectively [120, 121].
Shigella flexneri expresses a protein named IpaH9.8 that degrades the NF-kB essential modulator (NEMO) through its E3 ligase activity. This leads to disruption of NF-kB mediated inflammatory pathway . Microorganisms also use Skp1-Cul1-F-box (SCF)-type ubiquitin ligase to subvert the immune responses . For example, Vpr and Vif proteins encoded by HIV, target IRF3 for degradation presumably by the SCF complex .
Pathogens express not only PAMPs but also certain others factors that disrupt the immune response against them. These factors exploit the adaptor complexes but sometimes use TLR signaling to impede immune cell maturation . Cell wall component of mycobacterium tuberculosis contain mannose capped lipoarabinomannan (ManLAM) that induces IL-10 production leading to impairment in DC maturation . Mannose-containing ligands such as ManLAM trigger DC-SIGN to activate v-raf-leukemia viral oncogene 1 (Raf1), leading to acetylation of p65, which prolongs p65 DNA binding and enhances IL-10 transcription . Interestingly, in contrast to mannose, fucose containing ligands such as Lewis X, from Helicobacter pylori, dissociate Raf1-containing signal complexes and enhance IL-10 expression but downregulate IL-12 expression (in the case of mannose containing-ligands, IL-12 is also up-regulated). Thus, DC-SIGN ligands have various effects on the modulation of TLR-induced cytokine responses .