Toll-Like Receptors and Natural Killer Cells

Natural killer (NK) cells represent a heterogeneous subpopulation of lymphocytes of the innate immune system with a powerful antitumor activity, a function given by a complex collection of receptors. They act synergistically to recognize, regulate, or amplify the response according to the microenvironment, thus highlighting Toll-like receptors (TLRs), a type of receptors that allows sensing evolutionarily molecules conserved of pathogens known as pathogen-associated molecular patterns (PAMPs) and/or damage-associated molecular patterns (DAMPs). Those TLRs are essential to start the immune response. There is little information about the different subpopulations that form NK cells as well as their expression profile of innate immune response receptors in hematological cancers.


Introduction
Natural killer (NK) cells represent a highly specialized subpopulation of lymphocytes that are part of the innate immune system, whose functions vary according to the microenvironment. NK cells are involved in the early defense against foreign cells or own cells subjected to some stress (bacterial infection, viral or tumor transformation) through the recruitment of neutrophils, macrophages, dendritic cells, or B/T lymphocytes. They induce an effective adaptive response; regulate, directly or indirectly, the activity of antigen-presenting cells (APCs); and activate T lymphocytes through the natural cytotoxic activity that characterizes them or through the production of cytokines and chemokines that generate an inflammatory environment [1,2].
NK cells play an important role in the surveillance and suppression of tumor cells; despite the significant advances that have been reached in the last decades, it is still unknown if there is a direct relationship among the population dynamics, functionality, and the phenotype of these cells. Its role in the establishment and development of malignant hematological disorders such as acute lymphoblastic leukemia (ALL), a disease characterized by the uncontrolled proliferation of B or T lymphoid precursors, is still unknown.  (1) CD56 Bright CD16 Neg , recognized for its immunoregulatory activity, represents between 50 and 70% of the CD56 Bright population; (2) CD56 Bright CD16 Dim represent between 30 and 50% of the CD56 Bright population; (3) CD56 Dim CD16 Neg ; (4) CD56 Dim CD16 Bright is recognized for its cytotoxic activity; and (5) CD56 Neg CD16 Bright whose function is still unknown. Modified from [23].  [23]; they are mainly characterized by their poor cytotoxic capacity and their high capacity to secrete several types of post-activation cytokines, mainly INF-γ but also TNF-β, IL-5, IL-10, and IL-13 [7,8] and constitutively some chemokines such as MIP-1α, MIP-1β, and RANTES [24]. They also have the ability to proliferate in culture when exposed to low doses of IL-2 (picomoles) compared to the cytotoxic NK cells, which does not show an evident proliferation under the same conditions [25]. The NK Bright cells [CD56 High CD16 Neg ] are assigned an exclusively regulatory function, since their cytotoxic activity is poor and their genetic profile is directed mainly to the production of cytokines and not to the cytotoxic activity, in comparison with the NK Dim , whereas the NK Bright subpopulation [CD56 High CD16 Low ] is considered more as a transition phenotype [26]; because despite having the same characteristics of the previous phenotype, it has a lower rate of cell division when stimulated with IL-2 and does not change its activity even with ligands for c-KIT [27]. It exhibits cytotoxic activity [28], and it has also been seen that it represents the highest percentage of NK cells in circulation in bone marrow transplants, until its normalization around the fourth month [29,30]. NK Bright cells are usually not found in peripheral blood, bone marrow, and spleen as they are mainly distributed in secondary lymphoid nodules (parafollicular zone of T cells) [31] and tonsils [32].

Origin and distribution of NK cells in humans. The precursors of NK cell precursor (NKP) can originate from hematopoietic progenitor cells (HSC) in the bone marrow or from early lymphoid progenitor (ELP). NK cells mature mainly in the bone marrow, although the immature NKP and NK (iNK) can recirculate among the liver, spleen, and lymphoid nodes as alternative maturation sites. Mature NK cells (mNK) that leave the bone marrow reach different organs through blood circulation where they reside and modify their phenotypic and functional characteristics.
On the other hand, the NK Dim population represents around 90% of peripheral blood NK cells; they have a phenotype [CD56 Low CD16 Neg ] whose main function has not been well established [23] and a main phenotype [CD56 Low CD16 High ] that exhibits potent cytotoxic activity. Although they are generally poor producers of cytokines [7,8,33], they tend to predominate in the spleen, peripheral blood, and bone marrow [32,34].
It is important to clarify that the behavior of each subpopulation, in terms of post-activation secretion of cytokines and chemokines, will depend largely on the stimuli they receive either by recognizing target cells (tumor or transformed) or responding to exogenous cytokines.
The cytotoxic activity exhibited by NK cells does not require prior sensitization to kill their target cells, since it is not dependent on the presentation of a specific antigen as in the case of CD8 + T cells [7,36] and can be mediated through membranolitic and/or apoptotic mechanisms (Figure 3).
The membranolitic mechanisms include the production of perforins, enzymes that when integrated into the cell membrane form a pore that allows water to enter and cause osmotic lysis [37]. In the past, it was believed that both NK Bright and NK Dim cells had similar levels of perforins [38]; however, more recent studies by flow cytometry indicate that NK Dim cells have at least 10 times more perforins than NK Bright ones [35].
Regarding apoptotic mechanisms, these can induce the death of the target cell through complex mechanisms that involve both death-inducing proteins and specific ligand-receptor interactions through one of the following routes:

Granzyme pathway
Granzymes are proteins capable of activating the apoptosis program [39] following two mechanisms. The first does not depend on the activity of caspase proteins and is mediated mainly by granzyme A, and this fraction is single-stranded DNA (ssDNA) and interferes in the repair of genetic material without producing cell lysis [40,41], while the second promotes the activity of caspase proteins and is mediated mainly by granzyme B [42].
The NK cell presents granzymes A, B, K, and M; NK Dim cells present a high expression of granzymes A and B, whereas NK Bright cells mainly express granzyme K [35,43]. There are reports that NK cells express almost exclusively granzyme M; this enzyme is capable of mediating cell death independent of the activation of caspase proteins and in the presence of perforins, without fractionating DNA or producing changes in mitochondria [44].
In mice, deficient in granzymes A/B and/or perforins, it has been seen that there is uncontrolled growth of solid tumors, which suggests that these enzymes play an important role in the immunosurveillance of tumor cells mediated by NK cells [45].

NK cell ligand binding pathway to the cell death receptors expressed by the target cell
They include Fas ligands [FasL (CD95L)-Fas (CD95)] and/or the ligand that induces apoptosis related to tumor necrosis factor α (TRAIL) [46,47].

Antibody-dependent cellular cytotoxicity (ADCC)
NK cells express FcγRIIC/CD32c [48] and FcγRIIIA/CD16a [34]. These receptors interact with opsonized target cells, through the Fc regions of the antibodies, which combined with cellular antigens that cause the death of the target cell [49]. To through of mechanisms that involve the release of cytotoxic granules

Recognition and elimination of abnormal cells by NK cells. NK cells possess the ability to discriminate normal cells of tumor or transformed cells by detecting alterations at the HLA-I level; the target cells are eliminated by membranolitic and/or apoptotic mechanisms.
(perforin-granzyme), or by stimulation of apoptosis through of TNF-related apoptosis-inducing ligand (TRAIL) and/or by release of pro-inflammatory cytokines that promote the activity of other cells [50].
NK Dim cells with [CD56 Low CD16 High ] phenotype direct this mechanism in comparison with NK Bright cells. Although it has been seen that the subpopulation with [CD56 High CD16 Low ] phenotype exhibits low cytotoxic activity [51], NK Dim cells [CD56 Low CD16 Neg ] show a higher antitumor activity against cell lines (natural cytotoxicity) than other subpopulations [52]. This is supported by other studies where it is reported that NK Dim cells [CD56 Low CD16 High ] lose the expression of CD16 and increase the expression of CD107a (a degranulation marker), through a disintegrin and a metalloprotease-17 (ADAM-17), to become [CD56 Low CD16 Neg ] with high cytotoxic capacity [53].
The role of the [CD56 Neg CD16 High ] subpopulation is still not clearly defined. It is known that it is found in a low frequency in healthy individuals. It does not express surface molecules of other lymphoid lineages and that in chronic viral diseases, such as the human immunodeficiency virus (HIV). It presents changes in the level of expression of their activity receptors, characterized by the increase in the expression of inhibitory receptors and the decrease of natural cytotoxicity receptors (NCRs), together with other effector molecules that are hardly observed in healthy people [54][55][56].
It is considered that the [CD56 Neg CD16 High ] subpopulation is dysfunctional in terms of its lytic and antiviral activity, although it retains the ability to produce pro-inflammatory chemokines [54][55][56].
Zulu et al. demonstrated that the HIV induces the expansion of the negative CD56 population of NK cells through the upregulation of NKG2C receptors and the negative regulation of Siglec-7, NKG2A, and CD57 receptors [57].

Receptors of the NK cells
NK cells have signals through a wide variety of receptors that allow them to respond to different types of stimuli and grant great flexibility when exercising their effector and/or cytotoxic function.
The function of the NK cell is given by a complex collection of receptors that act in a synergistic way to recognize, regulate, or amplify the response according to the microenvironment. Thus highlighting the pattern recognition receptors (PRRs), such as Toll-like receptors or natural cytotoxicity receptors, and inhibitory killing receptors (iNKRs), such as receptors that are activated during early response to pathogens, cells transformed by virus or tumor cells [58].
PRRs are a family of innate immune response receptors that recognize evolutionarily conserved microbial products whose activation favors the production of pro-inflammatory cytokines. Within the PRR group, the TLRs are the most studied, although they are not the only ones; there are also the NOD-like receptors (NLRs) and the retinoid acid-inducible gene I (RIG-I)-like receptors (RLRs) [59].
NK cells express innate immune response receptors, such as NOD2, NLRP3, TLR3, TLR7, and TLR9, and promote the production of inflammatory cytokines and chemokines that are capable of amplifying the immune response [60]. The modulation of these cells through their innate immune response receptors, mainly via TLR, has gained interest and represents a promising therapeutic alternative against conditions such as cancer. Since there have been studies for a long time that support the possibility of its use, it has been observed that when ODNs (ligands of TLR9) are intraperitoneally administered in lymphoma murine models, an effective elimination of tumor cells occurs in 80% of cases [61].

Toll-like receptors and their role in NK cells
Toll-like receptors are among the most important group of pattern recognition receptors, since they orchestrate a wide variety of activities related to the immune response.
These receptors recognize a wide variety of molecules evolutionarily conserved, associated with microorganisms, such as lipopolysaccharides, lipoproteins, mycolic acids, non-methylated DNA, and double-stranded RNA, generically known as pathogen-associated molecular patterns [62][63][64]. TLRs also recognize endogenous molecules called damage-associated molecular patterns, which originate from damaged cells [65] or are products of altered metabolism of transformed cells in conditions such as cancer [66,67] and autoimmune diseases [67][68][69][70] or associated with chronic inflammation [71,72]. They play an important role in the evolution of these conditions.

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 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.

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.
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  , 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].  [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.

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.

Conflict of interest
The authors declare that there is no conflict of interest. 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]. © 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, provided the original work is properly cited.