**6. Epigenetics in natural killer cells**

**4.1. Functions of natural killer cells**

**4.3. Cytokine and chemokine release**

course after activation [92].

**4.4. Co-stimulation**

**4.2. Cytotoxicity**

38 Natural Killer Cells

and (C) co-stimulation of other immune cells.

In the immune system, the NK cells are of crucial importance due to its important functions that can be classified into three categories: (A) cytotoxicity, (B) cytokine and chemokine release,

Through their cytotoxic activity, NK cells are capable of killing viral infected and malignant cells [83]. Different cytotoxic pathways in NK cell have been characterized. First, the Prf- and Gzmmediated cytotoxic pathway is the most common killing pathway of NK cell. In such pathway, the well-known effector molecules (perforin-1 and granzyme B) are exocytosed from their cytoplasmic granules in NK cells to the vicinity of the target cells (immunological synapse) [70]. Prf1, which is a pore performing protein, polymerizes forming pores in the phospholipid bilayer of the target cell facilitating the delivery of granzymes into the cytosol of the target cell; whereas, the serine protease GzmB cleaves several procaspases and other intracellular substrates to initiate the classical apoptotic pathways [84]. Second, the killing process may also be mediated in a perforin-independent manner through the CD95 (Fas)-CD178 (Fas ligand) pathway. Upon binding of the NK cell to the Fas expressing stressed target cells, death inducing signaling complex is formed and subsequent activation of caspases promoting the apoptotic process of the target cell [85]. Third, the antibody-dependent cellular cytotoxicity (ADCC) which is used by leukocytes that express CD16 (Fc receptors), including NK cells to kill antibody-coated target cells [86].

It is thought that NK cells participate in a complex interaction network with other lymphocytes, dendritic cells, and macrophages to effectively control infection and malignancy. NK cells mediate their modulatory function on the immune cells through the production of cytokines and chemokines following either cytokine- or activating-receptor stimulation on the NK cell surface. The prototype effector cytokine produced by NK cells is IFN-γ, which has pleotropic effector actions on other immune cells, antigen-presenting cells, and virally infected or malignant target cells [7]. In viral models of infection, IFN-γ production by NK cells has been shown to be a key event in successful resolution of infection [87]. In liver, the IFN-γ contributes to the anti-viral [88], anti-fibrotic [89], anti-regenerative [90], and anti-tumor [91] activities of resident NK cells there. NK cells release several other cytokines including interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF), tissue necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β) and chemokines (MIP-1α, MIP-1β, and RANTES), which depends on the NK cell stimulation type and the time

NK cells can also interact with other immune cells through contact-dependent cell co-stimulation, this takes place upon the expression of several co-stimulatory ligands on the NK cell NK cells may show adaptive characters similar to B- and T-lymphocytes expressed in improved longevity and memory responses [103–105].

NK cells function mainly by producing interferon (IFN)-γ to eliminate pathogens, and release perforin to kill activated target immune cells [74, 106].

Epigenetics is a set of genome modifications that impacts the expression of a gene without changing the nucleotide sequence. Those modifications include methylation, acetylation, or phosphorylation of the histone proteins [107].

### **6.1. Epigenetic regulation of mature natural killer cell function**

The activation of NK cell has been studied for several decades; however, the mechanism of generation and maintenance of functional NK cells is only partially understood.

NK cell cytotoxicity is mediated by the effector molecule, perforin, which creates pores in the phospholipid bilayer of host target cells leading to facilitation of granzymes entry resulting in induction of apoptosis.

The expression of perforin in NK cells is only induced by the direct binding of myeloid Elf1-like factor (MEF), a transcription factor that belongs to Ets family, to perforin 1 promoter at two sites. However, in cytotoxic T lymphocytes perforin 1 is regulated differentially at the transcriptional level [108]. Perforin 1 regulatory region has two enhancers that are responsive to IL-2R-activated signal transduction and bind to Stat5 [108]. IL2Rb or Stat5b knockout NK cells of mice showed significantly lower levels of perforin transcript which highlights the importance of these enhancers for perforin transcription [109]. One of the enhancers is responsive to IL-6 and IL-12 and, upon cytokine stimulation, can bind STAT1a and STAT4, respectively [110, 111].

On the other hand, granzymes are serine proteases present in cytolytic granules of NK cells and cytotoxic T cells. Granzyme B (GZMB) is the most comprehensively studied member of the granzymes family. It acts on target cell either through cleaving caspases or damaging mitochondria thus mediating cell death [112]. Transcriptional regulation of granzyme B in CD8+ T cells is mediated by binding of transcriptional factors as CREB1, RUNX1, and AP1 to granzyme B promoter, harboring a DNase-hypersensitive region, enhancing the transcription [113–115]. Granzyme B expression is epigenetically controlled by histone H3K9 acetylation in its promoter resulting in high gene expression levels [113]. Another regulator of granzyme B expression is NF-κB, where, the activation of NF-κB signaling pathway results in binding of NF-κB to an enhancer element downstream to granzyme B transcriptional start site inducing its expression [116–118].

#### **6.2. Recent approaches in fine-tuning of natural killer cell function**

In a study conducted by our research group, a potential role of insulin-like growth factor-1 (IGF-1) was highlighted in modulating cytolytic potential of NK cells of HCC patients. miR-486-5p acts in a cell-specific manner, differentially modulating IGF-1 expression in NK cells and their target hepatocytes with a contemporary inhibitory impact on HCC progression [119]. Moreover, in another study, NK cells of HCC patients showed miR-182 overexpression compared to controls. NKG2D and NKG2A were upregulated and downregulated, respectively, in HCC NK cells. Upon forcing miR-182 expression in the HCC NK cells, upregulation of both receptors was observed. Finally, miR-182 was reported to induce NK cell cytotoxicity represented in perforin-1 upregulation and increase in cytolytic killing of co-cultured Huh-7 cells [120].

Our research team reported a novel role of miR-615-5p in NK cells activity of HCC patients. We have previously described miR-615-5p as potent tumor suppressor microRNA in HCC via repressing a pivotal mitogen in HCC, Insulin-like growth factor-II (IGF-II) as well as cellular proliferation, viability, and migration [121]. Recently, our team proved an opposing function for miR-615-5p in the NK cells of HCC patients. Forcing the expression of miR-615-5p repressed insulin-like growth factor-type 1 receptor (IGF-IR), attenuated NKs cytotoxicity, decreased CD56dim, increased CD56bright NK subsets, and reduced the cytotoxic markers NKG2D, TNF-α, and perforins. It also repressed NKG2D ligand (ULBP2) in Huh-7 cells [122].
