**2.8 Overview of the toll-like receptors**

Structurally, TLRs are type I integral glycoproteins that present an extracellular domain with leucine-rich repeats (LRRs) that are responsible for binding and discriminating ligands (PAMPs or DAMPS) present in the cellular microenvironment. They have a transmembrane domain and an intracellular Toll/interleukin (IL)-1 receptor (TIR) domain that triggers the signaling cascade via MyD88/TRIF and is highly conserved among each subfamily of TLRs [73].

There are 13 TLRs described in mammals, and 10 are found at the protein level in humans and differ according to their cellular localization and to the different PAMPs/DAMPs to which they respond. TLR11 in humans is a pseudogene, so it is not expressed [74].

The TLRs that are found mainly in the cell membrane are TLRs 1, 2, 4, 5, and 6. They sense structural components of bacteria, fungi, helminthes, or protozoa, whereas TLRs that are mainly found in intracellular compartments, such as TLRs 3, 7, 8, and 9, sense nucleic acids of viral and/or bacterial origin [73, 75] (**Figure 4**).

The stimulation of the TLRs is capable of initiating an immune response to various stimuli on its own, as well as of controlling the adaptive response through the inflammatory process with the production of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α), chemokines (IL-8, MCP-1) [76]; defensins [77]; type I interferons [78]; co-stimulation and MHC molecules [79]. The union between the different types of immune responses through the TLRs takes as a classic example the dendritic cells. They inspect their environment through the TLRs [80]; and once they detect a ligand (bacterial product, viral or stress protein), increase the expression of co-stimulatory molecules capable of stimulating T naive cells [81] and polarizes the adaptive immune response toward Th1 or Th2 profiles [82].

The cellular response through direct stimulation with TLR ligands will depend largely on the type of lineage in question. The information that is reported about activity and expression in NK cells is relatively new, and it is more associated with innate antibacterial or antiviral immune response [83], but not in cancer.

### **2.9 Expression of TLRs in NK cells**

The expression profile of TLRs in NK cells was initially limited to the detection of mRNA. However, the results do not always reflect the expression of the protein since it is difficult to identify the receptors in the NK cell.

NK cells express most of the human TLRs reported to date, although the detection and level of mRNA expression of each receptor vary depending on the author.

#### **Figure 4.**

*Toll-like receptors and their ligands. TLRs are transmembrane proteins of a glycoprotein nature that possess the ability to sense highly conserved microorganism molecules known as pathogen-associated molecular patterns (PAMPs), such as flagellin, LPS, or genetic material (ssDNA, ssRNA, dsDNA, dsRNA). In humans, 10 of the 13 TLRs present in mammals have been detected [84].*

There are authors who indicate that NK cells express high levels of TLR1, followed by TLR2, TLR3, TLR5, TLR6, and low levels of TLR4 [85, 86]. Other authors agree that TLR2 and TLR3 have greater expression, followed by TLR5 and TLR6; in those studies, TLR1 mRNA was not quantified [87–89]. On the other hand, TLR7 [85, 89, 90] and TLR10 [83, 86] present levels so low that they are practically undetectable.

There is a controversy about whether or not NK cells express TLR8 [86, 87, 89] and TLR9 [86, 88], although some authors point out that the cells constitutively express mRNA of all TLRs [85, 91, 92].

NK cells have higher levels of TLR3 mRNA than any other peripheral blood mononuclear cell, such as monocytes, B and T lymphocytes, or plasmacytoid dendritic cells [85].

Through techniques such as flow cytometry, Western blot, and immunoprecipitation, it is possible to know that NK cells of healthy people have a defined TLR expression profile (**Table 1**) and the expression of receptors is independent of their activation state [58].

In addition, there are variations in the level of TLR expression within the same subpopulations of NK cells. It is accepted that both NK**Bright** and NK**Dim** exhibit a similar mRNA profile of TLRs, although it is not always reflected at the protein level and there is a great controversy regarding the distribution and presence of some of these receptors in both subpopulations, especially TLR2, TLR4, and TLR3.

TLR2 and TLR4 are mainly distributed on the cell surface, whereas TLR3 is generally found in intracellular vesicles [75]; however, it has been seen that in NK cells, TLR3 is expressed both within [93] and on the cell surface [94]. There are publications reporting that TLR2 and TLR4 exhibit a marked intracellular distribution [95], although other authors indicate otherwise [96, 97].

The relative amount of some TLRs may vary according to the phenotype (**Dim** or **Bright**), although expression levels appear to be higher in cells with regulatory

**25**

*Toll-Like Receptors and Natural Killer Cells DOI: http://dx.doi.org/10.5772/intechopen.86393*

**TLR mRNA1 Protein Detection method**

**Presence** 1 Very high Yes Flow cytometry [97]

3 High/moderate Yes Flow cytometry [93, 94]

7 Very low/undetectable Yes Flow cytometry [93, 94]

9 Low2 Yes Flow cytometry [93, 95, 100]

*In previous studies, TLR2 could not be detected by flow cytometry or by immunoprecipitation.*

*More recent studies indicate that it is expressed mainly as intracellular [95, 99].*

*to flagellin [88, 101, 102], a molecule that it is only recognized through this receptor.*

8 Low2 Yes Flow cytometry [94]

5 High/moderate Yes5 S/R

10 Very low/undetectable N/R N/R

*the comparison of expression among the 10 TLRs.*

*Expression of TLRs in human natural killer cells.*

4 Low Yes4 Flow cytometry [94, 95, 99]

2 High/moderate Yes3 Direct activation of the TLR2-/MyD88-

6 High/moderate Yes Flow cytometry and Western blot [97]

Western blot [97]

Western blot

Western blot [94]

Western blot [94]

Western blot [90, 94]

Western blot [100]

*The levels of relative expression are given according to what was reported by [85, 91] and refer to* 

dependent pathway [96] Flow cytometry [95–97]

phenotype [89], which suggests that the type of response could be conditioned to promote a cytotoxic or immunomodulatory response when using one ligand or

It has been seen that NK**Bright** cells express more TLR1, TLR2, and TLR6 than NK**Dim** [97], although other studies report that less than 1% of total NK cells express

*There is a controversy whether or not they express mRNA of these TLRs, since some reports indicate that it was not* 

*No reports were found indicating the presence of TLR5; however it is inferred that it is present as it responds specifically* 

NK**Dim** cells can express, under normal conditions, more TLR4 than NK**Bright** cells [99] although other authors seem to find no differences in the expression in TLR2,

There is no information about whether there is differential expression of TLRs 3, 5, 7, or 8, and the distribution pattern of TLR5 is not known. However, it is inferred that NK cells express it, since they respond to flagellin and there are several studies that demonstrate it [88, 101, 102]. To date there are no reports about the presence,

The therapeutic use of TLR ligands in the modulation of NK cells against cancer,

especially in malignant hematological disorders such as leukemia, is an interesting alternative for the treatment of this type of diseases, since there are reports that reveal their therapeutic use as potential antitumor agents and as adjuvants in vaccines and other therapeutic modalities [103]. It is currently the subject of an

another in TLR activation assays (**Table 2**).

distribution, or role of TLR10 in NK cells.

extensive review by several research groups [104, 105].

these three receptors [98].

*N/R, not reported.1*

*possible to detect it.*

*2*

*3*

*4*

*5*

**Table 1.**

TLR4 [95], and TLR9 [95, 100].

#### *Toll-Like Receptors and Natural Killer Cells DOI: http://dx.doi.org/10.5772/intechopen.86393*

*Toll-like Receptors*

There are authors who indicate that NK cells express high levels of TLR1, followed by TLR2, TLR3, TLR5, TLR6, and low levels of TLR4 [85, 86]. Other authors agree that TLR2 and TLR3 have greater expression, followed by TLR5 and TLR6; in those studies, TLR1 mRNA was not quantified [87–89]. On the other hand, TLR7 [85, 89, 90] and TLR10 [83, 86] present levels so low that they are practically undetectable. There is a controversy about whether or not NK cells express TLR8 [86, 87, 89] and TLR9 [86, 88], although some authors point out that the cells constitutively

*Toll-like receptors and their ligands. TLRs are transmembrane proteins of a glycoprotein nature that possess the ability to sense highly conserved microorganism molecules known as pathogen-associated molecular patterns (PAMPs), such as flagellin, LPS, or genetic material (ssDNA, ssRNA, dsDNA, dsRNA). In humans, 10 of the* 

NK cells have higher levels of TLR3 mRNA than any other peripheral blood mononuclear cell, such as monocytes, B and T lymphocytes, or plasmacytoid

Through techniques such as flow cytometry, Western blot, and immunoprecipitation, it is possible to know that NK cells of healthy people have a defined TLR expression profile (**Table 1**) and the expression of receptors is independent of their

In addition, there are variations in the level of TLR expression within the same subpopulations of NK cells. It is accepted that both NK**Bright** and NK**Dim** exhibit a similar mRNA profile of TLRs, although it is not always reflected at the protein level and there is a great controversy regarding the distribution and presence of some of

these receptors in both subpopulations, especially TLR2, TLR4, and TLR3.

tion [95], although other authors indicate otherwise [96, 97].

TLR2 and TLR4 are mainly distributed on the cell surface, whereas TLR3 is generally found in intracellular vesicles [75]; however, it has been seen that in NK cells, TLR3 is expressed both within [93] and on the cell surface [94]. There are publications reporting that TLR2 and TLR4 exhibit a marked intracellular distribu-

The relative amount of some TLRs may vary according to the phenotype (**Dim** or **Bright**), although expression levels appear to be higher in cells with regulatory

express mRNA of all TLRs [85, 91, 92].

*13 TLRs present in mammals have been detected [84].*

dendritic cells [85].

**Figure 4.**

activation state [58].

**24**


*N/R, not reported.1 The levels of relative expression are given according to what was reported by [85, 91] and refer to the comparison of expression among the 10 TLRs.*

*2 There is a controversy whether or not they express mRNA of these TLRs, since some reports indicate that it was not possible to detect it.*

*3 In previous studies, TLR2 could not be detected by flow cytometry or by immunoprecipitation.*

*4 More recent studies indicate that it is expressed mainly as intracellular [95, 99].*

*5 No reports were found indicating the presence of TLR5; however it is inferred that it is present as it responds specifically to flagellin [88, 101, 102], a molecule that it is only recognized through this receptor.*

#### **Table 1.**

*Expression of TLRs in human natural killer cells.*

phenotype [89], which suggests that the type of response could be conditioned to promote a cytotoxic or immunomodulatory response when using one ligand or another in TLR activation assays (**Table 2**).

It has been seen that NK**Bright** cells express more TLR1, TLR2, and TLR6 than NK**Dim** [97], although other studies report that less than 1% of total NK cells express these three receptors [98].

NK**Dim** cells can express, under normal conditions, more TLR4 than NK**Bright** cells [99] although other authors seem to find no differences in the expression in TLR2, TLR4 [95], and TLR9 [95, 100].

There is no information about whether there is differential expression of TLRs 3, 5, 7, or 8, and the distribution pattern of TLR5 is not known. However, it is inferred that NK cells express it, since they respond to flagellin and there are several studies that demonstrate it [88, 101, 102]. To date there are no reports about the presence, distribution, or role of TLR10 in NK cells.

The therapeutic use of TLR ligands in the modulation of NK cells against cancer, especially in malignant hematological disorders such as leukemia, is an interesting alternative for the treatment of this type of diseases, since there are reports that reveal their therapeutic use as potential antitumor agents and as adjuvants in vaccines and other therapeutic modalities [103]. It is currently the subject of an extensive review by several research groups [104, 105].


*N/R, not reported.<sup>1</sup> It was found that both, in cell lines (NKL, NK92, and YT) and in NK of peripheral blood, TLR3 is expressed on the surface [94].*

*2 Studies that are more recent indicate that it is mainly expressed as intracellular [95, 99].*

*3 TLR9 expression exists in plasma membrane, but it is quite low compared to intracellular expression. 4 There are no studies that determine whether there is differential expression, although it has been seen that NKBright cells are better activated with ssRNA40 than NKDim cells, suggesting that the latter have a lower expression of TLR8.*

*5 In other studies, it seems that NKDim cells express more TLR9 than NKBright cells and its expression conditions the response to ligands of this TLR [100].*

#### **Table 2.**

*Localization and differential distribution of TLRs in human natural killer cells.*

### **3. Conclusion**

In this chapter, we included the overview of NK cells, their population diversification and role in the immune response, and their expression and role of TLRs.

#### **Acknowledgment**

We thank doctors Jacques Zimmer, MD, PhD, and Sankar Ghosh, PhD, for allowing us to use some of their figures, **Figures 2** and **4**, in this chapter of the book.

**27**

**Author details**

provided the original work is properly cited.

Federico Gómez, Mexico City, Mexico

Carmen Maldonado-Bernal\* and David Sánchez-Herrera

\*Address all correspondence to: cmaldobe@yahoo.com

*Toll-Like Receptors and Natural Killer Cells DOI: http://dx.doi.org/10.5772/intechopen.86393*

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Immunology and Proteomics Research Laboratory, Children's Hospital of Mexico

#### **Conflict of interest**

The authors declare that there is no conflict of interest.

*Toll-Like Receptors and Natural Killer Cells DOI: http://dx.doi.org/10.5772/intechopen.86393*

*Toll-like Receptors*

3 Intracellular [93] and extracellular1

4 Extracellular [94] and intracellular2

9 Mainly intracellular3

**3. Conclusion**

*N/R, not reported.<sup>1</sup>*

*2*

*3*

*4*

*TLR8. 5*

**Table 2.**

*is expressed on the surface [94].*

*response to ligands of this TLR [100].*

**Acknowledgment**

**Conflict of interest**

book.

In this chapter, we included the overview of NK cells, their population diversifi-

**TLR Cellular localization Population distribution** 1 Extracellular [97] NK**Bright** > NK**Dim** [97] 2 Extracellular [96, 97] and intracellular [95] NK**Bright** > NK**Dim** [97]

5 N/R N/R 6 Extracellular [97] NK**Bright** > NK**Dim** [97]

7 Intracellular [93, 94] N/R 8 Intracellular [94] N/R4

10 N/R N/R

*TLR9 expression exists in plasma membrane, but it is quite low compared to intracellular expression.*

*There are no studies that determine whether there is differential expression, although it has been seen that NKBright cells are better activated with ssRNA40 than NKDim cells, suggesting that the latter have a lower expression of* 

*In other studies, it seems that NKDim cells express more TLR9 than NKBright cells and its expression conditions the* 

*Studies that are more recent indicate that it is mainly expressed as intracellular [95, 99].*

*Localization and differential distribution of TLRs in human natural killer cells.*

[94] N/R

[93, 95, 100] NK**Bright** = NK**Dim**<sup>5</sup>

*It was found that both, in cell lines (NKL, NK92, and YT) and in NK of peripheral blood, TLR3* 

[95, 99] NK**Bright** < NK**Dim** [99]

NK**Bright** = NK**Dim** [95]

[95]

cation and role in the immune response, and their expression and role of TLRs.

We thank doctors Jacques Zimmer, MD, PhD, and Sankar Ghosh, PhD, for allowing us to use some of their figures, **Figures 2** and **4**, in this chapter of the

The authors declare that there is no conflict of interest.

**26**
