TLR10: An Intriguing Toll-Like Receptor with Many Unanswered Questions Open Access

Prior to the discovery of Toll-like receptors (TLRs), innate immunity was thought to be a simple and undeveloped component of the immune system that triggered a more complex adaptive immune response to, only then, protect the infected organism [1]. However, the characterisation of TLRs has allowed the identification of other families of innate immune receptors, providing immunologists new insights on the innate immune response.

First described as a transmembrane protein of Drosophila melanogaster embryos, TLRs are a type of pattern recognition receptor that connect the innate and adaptive responses [3]. By showing that mutations in the Toll signalling pathway dramatically reduced Drosophila’s survival after fungal infection, Lemaitre et al. [4] discovered in 1996 that the Toll protein’s function was associated with immunity. In 1998, five Toll homologues, including hToll (renamed as TLR4), were described and called TLRs [5]. Ten human and 12 mouse TLRs are known to date [6]. They can be expressed on the plasma membrane (TLR1, TLR2, TLR4, TLR5, TLR6, and TLR10) or in the intracellular vesicles, such as endosomes, lysosomes, and endolysosomes (TLR3, TLR7, TLR8, and TLR9) [8]. The plasma membrane TLRs play a critical role in the recognition of few evolutionarily conserved microbial molecules present on extracellular pathogens (PAMPs), while intracellular TLRs mainly recognise pathogen nucleic acids and sometimes damage-associated molecular patterns [8]. Thus, the host defences are adapted according to the pathogen’s lifestyle inside the host, which results in the activation of various transcriptional and nontranscriptional responses and the recruitment of specific immune cells [10].

After the recognition of PAMPs and damage-associated molecular patterns, TLRs recruit proteins known as myeloid differentiation primary response protein 88 (MyD88), TIR domain-containing adaptor protein (TIRAP), TRIF-related adaptor molecule (TRAM), and TIRAP-inducing IFN-β (TRIF), which will trigger signal transduction pathways, activate transcriptor factors such as NF-κB, IRFs, and MAP kinases to control the expression of proinflammatory chemokines, type I IFN, and cytokines and protect the host against microbial infection [6]. Apart from TLR3, all other TLRs attract MyD88 and start MyD88-dependent signalling, which activates NF-κB and MAP kinases and causes macrophages and dendritic cells to produce proinflammatory cytokines. Both TLR3 and endosomal TLR4 use TRIF, which activates the expression of type 1 IFN without MyD88 (MyD88-independent) [11]. The different PAMPs recognised by the TLRs are displayed in Table 1 and were analysed in previous reviews [8, 12‒14]. The most recent TLR to be identified is TLR10 [15], and since its discovery, TLR10 has received increasing research interest. However, there is controversy on the function of TLR10 as an anti-inflammatory or proinflammatory TLR, and both its function and ligands remain to be further investigated.

TLR10 is a transmembrane glycoprotein composed of an extracellular, a transmembrane, and a cytoplasmic region [16]. Similar to other plasma membrane TLRs, TLR10 extracellular domain has a leucine-rich repeat, which is responsible for ligand interaction [17]. Despite having different evolutionary origins, plants also present leucine-rich repeat domains that are important for their bacterial, fungal, and viral resistance. The conservation of this domain suggests its relevance for the immune response against microbial pathogens [18]. The transmembrane region of TLR10 is also important for immune responses as it can form homodimers of TLR10 or heterodimers between this receptor and other TLRs, such as TLR1 and TLR2 [20]. TLR10 interactions and function, especially those involving TLR2 and TLR10, are regulated by dimerisation [22].

In the article that first described TLR10, Chuang and J. Richard aligned the amino acid sequences of the cytoplasmic region of human TLRs and showed that 13 amino acids present in the C-terminal end of TLR2 are highly conserved in TLR10. According to the authors, these amino acids are necessary for intracellular downstream signalling cascades [15]. The intracellular region of TLRs contains the Toll/interleukin-1 receptor [23] domain, and it has been associated with the recruitment of MyD88, TRIF, MAL, TRAM, and TIRAP that will lead to NF-κB and IRF activation [24] (Fig. 1).

TLR10 can also be expressed in endosomes, where it has been shown to recognise dsRNA in vitro. In response to dsRNA recognition, TLR10 recruits MyD88 [25]. By using a recombinant transmembrane TLR10 protein, Hasan et al. [26] showed that it also directly associates with MyD88. However, whether this association can activate NF-κB has not been validated. Since its discovery, the lack of a mouse model has been a significant barrier to research on TLR10 signalling, as TLR10 is a pseudogene in mice.

Human TLR10 is mainly expressed in lymphoid tissues such as lymph node, thymus, tonsil, and spleen [27]. The genes for TLR1, TLR6, and TLR10 are in the same chromosome locus 4p14 [28] (Fig. 2). Furthermore, TLR10 shares 50% and 49% amino acid identity with TLR1 and TRL6, respectively [15], suggesting a close relationship between these receptors. However, unlike TLR1 and TLR6, TLR10 is expressed as a highly N-glycosylated protein that has been found in plasmacytoid dendritic cells from tonsils, B-cell lines, and peripheral blood B cells [26].

A previous study has shown that TLR10 expression increases as B cells become activated and mature [29]. This increase in expression suggests that TLR10 may play a role in regulating B-cell function and B-cell-related diseases. In fact, human TLR10 polymorphisms have been associated with a broad range of diseases, including bacterial infections, cancer, and autoimmune diseases [30‒32]. Zhang et al. [33] found a positive correlation between rheumatoid arthritis disease activity and increased TLR10 expression in B cells. On the contrary, Lai et al. [31] found that TLR10 expression was significantly downregulated in CD19+ B, naive B, and memory B cells of high-activity primary Sjogren’s disease (pSjD) patients compared to low-activity pSjD patients, indicating that TLR10 would prevent the progression of pSjD by inhibiting B-cell activation and the production of autoantibodies. These apparently contradictory data underscore the need for further studies on the expression of TRL10 in various disease conditions.

The identification of TLR10 biological ligands would benefit the understanding of its function. In this sense, several ligands have been proposed for TLR10, including flagellin and synthetic diacylated lipoprotein known as fibroblast-stimulating lipopeptide (FSL-1) [34].

Some known TLR2 ligands also bind to TLR10. LPS was found to be a potential ligand of TLR2/TLR10 heterodimer after Helicobacter pylori infection [35]. Regan and colleagues described the activation of NF-κB through the TLR2/TLR10 heterodimer after Listeria monocytogenes infection [36]. A more recent study also identified HIV gp41 as a ligand of TLR10, leading to NF-κBα activation and IL-8 production. Moreover, the authors showed that TLR2 and TLR10 each play a distinct role in the regulation of HIV-1 infection [37].

Ligand recognition and immune system modulation are increased by homodimer and heterodimer formation between TLRs, which could explain the wide variety of PAMPs detected for TLRs [38]. The TIR domain mediates TLR signalling [39]. The crystal structures of the TIR domains of TLR1, TLR2, and TLR10 presented a conserved fold of five parallel β-sheets encircled by five α-helical segments [40]. The association of two different TIR domains allows the recruitment of specific adaptor molecules, leading to appropriate immune-gene expression.

TLR10 has been found to act as a proinflammatory TLR as homodimer or as TLR10/TLR1 heterodimer [41]. While some studies have indicated that TLR10 inhibits TLR2, others have observed that it enhances TLR2 activity upon forming a dimer with TLR2 [35].

Although TLR10 can recognise TLR2 agonists, such as Pam3Cys and FSL-1, few studies have examined whether it might recognise TLR2 antagonists [34]. The interactions between TLR2 and TLR10 require additional investigation to be fully understood. In that context, research on the competitiveness of these proteins and on their TIR structure after dimerisation are needed to better comprehend their interconnection.

Although multiple studies have been conducted on TLR10 function, the results are inconclusive. TLR10 is expressed on Tregs, where its expression is regulated by Forkhead box P3 (FOXP3), indicating that TLR10 has a different function from other TLRs [45]. Hess and colleagues showed that the typical proinflammatory signalling pathways can be suppressed by antibody recognition of TLR10, leading to a reduction in proinflammatory cytokine levels. Additionally, the authors showed that by reducing the capacity of dendritic cells to activate T cells, the antibody recognition of TLR10 can affect the differentiation of monocytes into dendritic cells [46]. TLR10 knockdown in a human monocyte cell line resulted in a reduction in the expression of inflammatory cytokines such as IL-8, IL-1, and CCL20 induced by FSL-1, LPS, and flagellin [34]. Another study using a different monoclonal anti-TLR10 antibody demonstrated suppression of LPS-induced release of proinflammatory cytokines [47]. These results suggest that TLR10 can act as a negative regulator of other TLRs by controlling immune responses.

However, other studies have shown TLR10 involvement in the upregulation of inflammatory reactions. Neutrophils are the first immune cells to be recruited to acute inflammation sites. Using human neutrophils, we analysed TLR10 expression before and after LPS stimulation. Changes in TLR10 localisation from the plasma membrane to the cytoplasmic vesicles were observed after 1 h of LPS treatment. After 2 h, TLR10 reappeared in the cell membrane, suggesting that LPS induces TLR10 raft-dependent endocytosis. Furthermore, our data showed that TLR10 expression was decreased by reactive oxygen species (ROS) depletion, TLR4 neutralisation, and NF-κB inhibition, indicating that these factors may play a significant role in TLR10 regulation [48].

In accordance with our findings, Sindhu et al. [49] showed that TLR10 expression is increased by oxidative stress and that it may serve as an immunological marker for metabolic inflammation. A previous study analysed the consequences of ROS and hypoxia on TLR10 expression, revealing that both hypoxia and ROS can upregulate TLR10 expression by NF-κB activation in a THP-1 myelomonocytic cell type [50]. Another study used the same cell line and demonstrated that H. pylori significantly increases TLR10 mRNA in a time-dependent manner after 6 and 24 h of the bacterial treatment [51]. Lee et al. [38] demonstrated that cellular responses to H1N1 and H5N1 influenza viruses were reduced by TLR10 knockdown, indicating the contribution of this receptor to microbial illnesses.

It has been suggested that the TLR1-TLR6-TLR10 gene cluster is a hotspot for positive selection since it was also positively selected independently in the genomes of apes as chimpanzees and orangutans [52]. This selective sweep may also point to relevant functional effects of genetic variation of the TLR1-TLR6-TLR10 locus. Indeed, genetic variations in this locus have been shown to alter the expression of all three TLRs and cause vulnerability to immune-mediated illnesses and allergies [53]. Although it is difficult to establish which of the three TLRs was targeted for positive selection, some studies have shown that TLR10 was the primary target [54]. These opposing findings imply that TLR10 modulatory effects are complex and that TLR10 may behave differently in response to pathogens or ligands, activating various signalling pathways or interacting with different pattern recognition receptors.

All known TLRs are expressed widely on both pulmonary system as well as primary immune cells which respond to a lung infection or inflammation. We earlier reported the expression of TLR10 in normal and inflamed lungs of cattle, pig, rat, and chicken, and normal lungs of dog [55]. Immunohistochemistry of control calf lung localised TLR10 in subepithelial area of bronchioles, subendothelial area of alveolar blood vessels and alveolar septa as well. TLR10 expression is increased in lungs from pigs infected with swine influenza virus, indicating the proinflammatory role of TLR10 during viral infection. Immuno-electron microscopy of cattle lung showed TLR10 in alveolar epithelium, vascular endothelium, cytoplasm, and nucleus of pulmonary intravascular macrophages (Fig. 3) [55]. Furthermore, immune-fluorescent staining with TLR10, Mac387, and vWF antibodies of paraformaldehyde-fixed human lungs showed punctate distribution of TLR10 on alveolar epithelium (unpublished data, indicated in Fig. 4) and overlapping staining of TLR10 with Mac387 (white arrows).

A pilot study on genetic association for analysing biologically probable candidate genes in 112 chronic cavitary pulmonary aspergillosis patients and 279 healthy controls showed the potential role of TLR3 and TLR10 in the pathology [56]. Single nucleotide polymorphisms (SNPs) of rs11466617 and rs412900 as well as a dose-dependent effect of TLR10 SNPs were shown to reduce the susceptibility to tuberculosis in Tibetans [57], indicating the role of TLR10 SNPs in tuberculosis pathogenesis.

Since TLR10 is a pseudogene in mice, there is a need for other experimental models to better comprehend this receptor. In this sense, several alternatives have been used to investigate TLR10’s potential role in vivo. By introducing the human TLR10 open reading frame into the ROSA26 locus, mice expressing human TLR10 were created [58]. Moreover, TLR10 is expressed in rats [59], making these animals a viable study model. Other alternatives that have been used in functional studies are human TLR10 knockdown immune cells [34], computational analysis, and human cell culture [35]. However, it must be emphasised that studying TLR10 in an in vivo model will be critical to determine physiological relevance of results obtained in vitro using cell lines.

Since their discovery in D. melanogaster, TLRs have been shown to have a role in immune and inflammatory diseases, as well as in cancer [60]. Advances in our understanding of the signalling pathways activated by TLRs, structural insights into the interactions between these receptors and their ligands, and methods to suppress TLRs are pointing to the potential use of these receptors as therapeutic treatment for inflammatory diseases.

It has been predicted that blocking TLR signalling can be an effective strategy for reducing inflammatory reactions. A variety of TLR antagonists and agonists have been developed to treat inflammatory diseases such as Alzheimer’s disease, sepsis, Crohn’s disease, Parkinson’s disease, and rheumatoid arthritis [60]. Inhibiting the intracellular signalling of the TLR pathways and blocking the binding of these receptors’ agonists are the two main methods of TLR inhibition [61].

Antibodies, microRNAs, nano-inhibitors, oligonucleotides, lipid A analogues, and small molecule inhibitors can be used as TLR inhibitors. MicroRNAs act on the intracellular signalling of TLR cascades, while antibodies, lipid A analogues, and oligonucleotides act on the ligand-receptor interaction. Small molecule inhibitors can suppress TLR signalling by both methods [61].

TLR10 can dimerise with itself or other TLRs, presenting pro- or anti-inflammatory functions (Fig. 5) [41]. Polymorphisms in the TLR10 gene may alter the ratio of pro- to anti-inflammatory responses, contributing to diseases such as malignancies and autoimmune disorders [31]. Furthermore, TLR10 signalling can change if its TIR domain is altered by mutations [39]. However, to target TLR10 for the treatment of disorders involving inflammatory or immunological responses, it is necessary to better comprehend its ligands, signalling, pathways of dimerisation, and activities. Understanding how TLR10 carries out its function can benefit the medical field since this will serve as a starting point for the therapeutic and diagnostic evaluation of illnesses that can be significantly impacted by TLR10 polymorphisms and signalling.

TLR10 is a transmembrane glycoprotein that is mostly expressed in immune cell-rich tissues [27]. Although the ligands and function of TLR10 are still unknown, contradictory data has been found regarding its proinflammatory/anti-inflammatory profile. Therefore, more research is necessary to answer the controversies. It is essential to comprehend the mechanism by which TLR10 carries out its role and its natural ligands. This understanding will allow further research into this distinct receptor, which can potentially be used as a therapeutic intervention against inflammation.

The authors have no conflicts of interest to declare.

The work was performed in the laboratories of Dr. Gurpreet Kaur Aulakh and Dr. Baljit Singh, and it was supported through grants from Natural Sciences and Engineering Research Council of Canada. Dr. Gurpreet Kaur Aulakh holds Sylvia Fedoruk Chair in Nuclear Imaging.

Dr. Carolina Rego Rodrigues and Dr. Yadu Balachandran performed the literature review and took the lead role in writing the written report. Dr. Baljit Singh and Dr. Gurpreet Kaur Aulakh reviewed and contributed to drafts of the written report.

Carolina Rego Rodrigues and Yadu Balachandran contributed equally to this work.