Beutler group identified TLR7 along with TLR8 and TLR9 within an established genomic database in the year 2000 (Du et al. 2000). TLR7 was evolved from common ancestral gene about 150 million years ago and was found to be X linked (Du et al. 2000). In 2002, synthetic chemical imiquimod was reported to be sense by TLR7, which was characterized as another antiviral TLR (Hemmi et al. 2002). Later in 2004, two groups independently identified TLR7 as a sensor of single-stranded viral RNA (Diebold et al. 2004; Heil et al. 2004). There are several synthetic compounds belonging to imidazoquinoline family, which have been identified as TLR7 and/or TLR8 agonists (Diebold et al. 2004). Among these agonists, the lead compound imiquimod exerts strong antiviral and antitumoral activities and is being marketed for the treatment of external genital warts caused by human papillomavirus (Wolf et al. 2003).
Pattern recognition receptors are major players of innate immune system. They are expressed by variety of immune cell types in both humans and mice. Some of them are present on the surface of the cell, while others are present within the cell (Kawai and Akira 2010). These receptors are evolutionarily conserved structures capable of recognizing microbe-associated molecular patterns, conserved in microorganism (Song and Lee 2012). The receptors on the surface are mostly known to recognize bacterial or fungal molecules, while those present inside the cell are more efficient in recognizing viral molecules and endogenously generated DAMPs (Kawai and Akira 2006, 2010). Intracellular localization of TLRs is important in preventing unnecessary activation upon coming in contact with self-nucleic acid molecules. Further these receptors are classified into four different classes as TLRs, CLRs, RLRs, and NLRs. Among the known TLRs, TLR3, TLR4, TLR7, TLR8, and TLR9 are present on the endosomes and are known to sense nucleotides. Except TLR3, all endosomal TLRs utilize MyD88 as an adaptor molecule, while TLR3 and TLR4 upon localized to endosomes use TRIF to control their downstream pathways. Here we are focusing on TLR7 and its role in various biological processes.
TLR7 is expressed both on the surface and on endosomes of the cells (Kanno et al. 2015) and recognizes genetic material from ssRNA viruses such as influenza A virus and HIV virus. Surface TLR7 activation with various TLR7 ligands was shown to induce a signaling cascade in a dose-dependent manner. On the other hand, blocking of TLR7 using the anti-TLR7-mAb results in the inhibition of downstream signaling leading to the abrogation of cytokine production in the various subsets of dendritic cells (Kanno et al. 2015)]. However, we believe that surface TLR7 do not induce a robust signaling, as it exists in its native form, while to initiate signaling cascade, its ectodomain has to undergo proteolytic processing between LRR14 and LLR15 (Z loop) (Ewald et al. 2008; Ewald et al. 2011). The presence of Z loop is a characteristic of TLR7, which allows it to recognize ssRNA, different from that of TLR8 (Zhang et al. 2016). Further, recognition of ssRNA by TLR7 is confirmed from experiments performed in TLR7-deficient mice, where the deficiency of TLR7 resulted in low levels of serum IFN-α after systemic ssRNA viral infection (Lund et al. 2004). Initially it was mentioned that TLR7 recognizes only viral RNA, but later on few studies have reported the recognition of RNA from bacteria translocated to the phagosomes (Mancuso et al. 2009). All three forms of bacterial RNA (mRNA, rRNA, and tRNA) are recognized by TLR7. Besides recognizing RNA molecules, TLR7 also responds to the oligonucl)eotide-guanosine (G) and its derivative (Shibata et al. 2016). A synergistic cooperation between short nucleotides and ssRNA is reported to be required for the activation of TLR7 (Zhang et al. 2016).
TLR7 Structure and Distribution
Among intracellular TLRs, TLR7 is located on endosomes, consisting of two different isoforms in mice that differ in amino acids (aa) residues from each other. Isoform (a) is shorter having 1050 aa residues than isoform (b), which has 1053 aa, three aa residues more than isoform (a). However in humans, TLR7 exist only in one form consisting of 1049 aa residues (Fig. 1). The ectodomain of TLR7 consists of 27 LRRs and is represented as shoe-shaped structure similar to other known TLRs (Matsushima et al. 2007). The predicted structures based on mathematical modeling showed that it contains leucine-rich repeats that are involved in the recognition of their cognate ligands. However, Zhang et al. have recently solved the crystal structure of TLR7 and showed the existence of two ligand-binding sites in its ectodomain for small ligand-binding molecule guanosine and ssRNA (Zhang et al. 2016). The first site preferentially binds guanosine and the second binding site recognizes uridine moieties present in ssRNA. Binding of ssRNA at the second site enhances the affinity of the first site for guanosine (Fig. 1b). The structural studies also reveal that in the absence of ligand, TLR7 exists as monomer.
The cells of both hematopoietic and nonhematopoietic origin express TLR7. In the hematopoietic compartment, based on gene analysis, its expression is reported in both lymphoid and myeloid cells. TLR7 expression in B-lymphocytes is reported to control antibody production. However, excessive interaction of ligand with TLR7 in B cells can result in autoimmune diseases (Pisitkun et al. 2006). On the other hand, in myeloid cell population, it is monocytes and pDCs that express very high level of TLR7 compared to other conventional DCs and macrophages. In tissues, TLR7 is mainly expressed in lung, brain, stomach, placenta, and peripheral blood mononuclear cells (Gentile et al. 2015).
TLR7 is a member of IL-1R/TLR superfamily containing cytoplasmic TIR domains (Akira and Takeda 2004). Unlike surface TLRs, it is present in the endosomes along with other TLRs sharing functional properties of recognizing nucleic acids, which are either translocated or are released within the endosomal compartments (Akira and Takeda 2004). Concomitantly, both TLR7 and TLR9 associate with the polytopic membrane protein UNC93B and are delivered to endolysosomes (Kim et al. 2008). Here they become competent for signaling after cleavage of the TLR ectodomain by lysosomal proteases. The mature TLR molecule contains an endolysosome-located ectodomain of leucine-rich repeats, a transmembrane domain, and a cytoplasmic domain known as the Toll/interleukin-1-receptor (TIR) domain. However, recently, TLR7 has been reported to present on the cell surface as well, which was previously unknown because of the lack of reagents (Zhang et al. 2016). The actual role of surface TLR7 expression could be attributed to endocytosis of ssRNA molecules, which then is translocated to the endosomes. But this report also then raises the concern of how self nucleic acids are not being recognized, which could cause severe problem. Additionally, a recent study by Means group showed that TREML4, a surface receptor, consists of extracellular immunoglobulin-like domain, a transmembrane domain, and intracellular cytoplasmic tail lacking signaling motif fine-tune TLR7-dependent immune responses (Ramirez-Ortiz et al. 2015). Absence of TREML4 partially abrogates TLR7 signaling and enhanced expression of it leads to more robust signaling by TLR7 (Ramirez-Ortiz et al. 2015).
Additionally, Myd88 recruited downstream of TLR7 also engage interferon regulatory element IRF7 through TRAF6 and IRAK4 (Guiducci et al. 2008). Ubiquitylated TRAF3 interacts with IRAK1/2 and IKKα to activate IRF7, which then translocates to the nucleus and initiates the transcription of type I IFN genes. The overall TLR7 signaling leads to robust pro-inflammatory program that helps in controlling bacterial and viral infections by upregulating co-stimulatory molecules and necessary signal 3 required for strong adaptive immune responses.
Role of TLR7 in Health and Diseases
The evolutionarily conserved pattern recognition receptor, TLR7, maintains homeostasis by differentiating foreign and self-molecules based on sequence, structure, and compartmentalization (Gehrig et al. 2012; Jöckel et al. 2012). Thus, recognition of microbial-derived nucleic acids by TLR7 initiates immune responses that help in their clearance (Mogensen 2009). However, recognition of self nucleic acids by B cells results in the production of autoantibodies, leading to the development of autoimmune diseases like systemic lupus erythromatosus (Green et al. 2009). Further in MRL-Fas lpr mice, TLR7 deficiency results in low serum titers of autoantibodies and less severe form of disease (Christensen et al. 2006). TLR7 also plays an important role in the development of diabetes as excessive stimulation produces interferon-α in pancreatic islet cells, which then promotes the development of autoimmune diabetes in NOD mice (Lee et al. 2011). The studies performed on mice deficient in TLR7 show delayed onset of diabetes, due to lower levels of pro-inflammatory cytokines and chemokines.
TLR7 importance has been demonstrated during viral infection, where its absence could lead to severe complications. The main cell types that respond to TLR7 stimulus (ssRNA from viruses) are pDCs and monocytes that help in the clearance of both viral and bacterial pathogen by producing interferon-α and by upregulating co-stimulatory molecules (Lund et al. 2004). However, study by Scott et al. showed that TLR7 is not required for the recognition of Influenza virus but is important for the induction of humoral immune responses (Jeisy-Scott et al. 2012). In contrast to influenza virus infection, studies with SIV or HIV-1 virus infection in human or macaque monkeys model showed the role of TLR7 in disease progression by inducing robust inflammatory responses (Wonderlich and Barratt-Boyes 2013). A study by Schott et al. showed that during chronic HCV-infection, TLR7 is detrimental as it promotes inflammation-associated liver fibrosis, while patients with single nucleotide polymorphism in TLR7 gene were found to be protected against HCV-induced inflammation and fibrosis (Schott et al. 2007). However, in a cancer model, stimulation of in pancreatic cancer cells results in increased tumor cell proliferation and reduced chemo-sensitivity.
TLR7 as a Therapeutic Agent
Agonists for TLR7 showed great potential as vaccine adjuvants by directly activating antigen-presenting cells and in the induction of both humoral and cell mediated immune responses. Synthetic molecules imidazoquinolines, such as imiquimod and resiquimod, are good source of TLR7 agonists and are used as vaccine candidates in treating genital warts and skin carcinomas (Wolf et al. 2003). As of to date, topical application of TLR7 is used to control basal cell carcinoma and actinic keratosis, but its systemic application does not show any promising result (Meyer et al. 2013). Additionally studies in Rhesus Macaque monkeys showed multiple oral administrations of TLR7 agonists suppress HIV virus-like infection in the absence of antiretroviral therapy, thus making a case for it to be a regimen for treating viral infection as well. However, multiple exposures to TLR7 agonists induce tolerance in experimental allergic encephalomyelitis murine model (Hayashi et al. 2009).
The recent advancement in the field of TLR7 biology has increased our understanding about its expression and the role of accessory molecules in controlling TLR7-dependent immunological responses. The surface expression of TLR7 might allow us to better understand the previously unanswered question of how exogenous ligand can be recognized by endosomal TLR7. Further examination is required to fully understand the role of surface TLR7 in the development of autoimmune diseases and in the induction of immune tolerance. Recent data showed that targeting of TREML4, a surface glycoprotein, could be useful in controlling TLR7-mediated immune responses. Thus, deciphering how TLR7-specific adaptor molecules control immune responses is an exciting and rapidly expanding field with important implications in both basic and translational immunology.
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