Nk Cells, Activating and Inhibitory Receptors in Influenza Virus Life Cycle

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Table of Contents

Natural killer (NK) cells
Missing-self hypothesis
Human NK cells
Murine NK cells
NK cell receptors
Influenza A virus
How influenza viruses can escape NK cell recognition?

Natural killer (NK) cells

NK cells were first discovered in 1975 as lymphocytes of the innate immune system that can kill leukemia cells in vitro without previous sensitization:

Since then, NK cells have been revealed to play an important role in the early defense against certain viruses, microbial infections and cancer
Recently, NK cells have been implicated in the regulation of adaptive immune responses following an inflammatory response through the elimination of specific antigen-activated T cells
In contrast to T and B cells, NK cells do not express rearranged antigen-specific receptors. This is because an alternative NK effector function is tightly controlled by the combination of signals received through germ-line-encoded receptors with inhibitory
Activating functions that can recognize ligands on their cellular targets.

Inhibitory receptors, such as the inhibitory Ly49, killer cell immunoglobulin-like receptors (KIRs), leukocyte immunoglobulin-like receptors (LILRs) and CD94-NKG2A receptors, bind to self-MHC class I, or MHC class-I like molecules and signal through immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in their cytoplasmic tail. Some of the transformed and virus-infected cells tend to down-regulate normal expression of MHC class-I in order to avoid recognition by CD8+cytotoxic T lymphocytes and that makes them super susceptible to NK cell-mediated killing. Activating NK receptors, such as NKG2D, KIR2DS2 and CD94-NKG2C are structurally related to inhibitory receptors but lack ITIMs. They also associate with signaling molecules such as DAP12, CD3ζ, or FcRγ, which signal through immunoreceptor tyrosine-based activating motifs (ITAMs), located in its cytoplasmic tail (5). Additionally, NK cells also express the low-affinity Fc receptor (CD16), thus enabling them to detect antibody-coated target cells and exert antibody-dependent cellular cytotoxicity (ADCC) (6).

NK cells kill their target cells by either of two mechanisms that require direct cell-cell contact. In the first pathway, cytoplasmic granule toxins are known as perforin (pore-forming protein) and granzymes (serine proteases) are secreted by exocytosis and together they induce apoptosis of the target cell. The second pathway, apoptosis via Fas/Fas ligand (FasL) interactions involves the engagement and aggregation of Fas-expressing target cells by its cognate ligand, FasL, which is expressed on NK cells’ cell membrane, resulting in apoptosis of the target cell (7).

Furthermore, upon stimulation, NK cells can secrete potent levels of cytokines especially interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), IL-10, IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), and chemokines such as CC chemokine ligand 3 (CCL3), CCL4 and CCL5 (8). Studies have also demonstrated a critical role of several cytokines in the induction of NK-cell cytotoxicity. These include interleukin-12 (IL-12), IL-18, IL-15, and type I interferons (9).

Studies have shown that IL-12 release by mature DCs leads to the production of IFN-g by NK cells, which increases the surface expression of MHC class I and MHC class II on surrounding cells enhance their recognition by cytotoxic T cells. Moreover, IFN-g further activates macrophages, dendritic cells, and enhances Th1 activation and polarization by inducing T-bet expression (10, 11).

Missing-self hypothesis

A hypothesis proposed by Klas Kärre and collaborators suggests that the absence or altered expression of MHC class I molecules on the cell surface of transformed or virus-infected cells renders these cells significantly susceptible to NK cell cytotoxicity. This observation led to the “missing-self” hypothesis, which proposed that NK cells recognize and destroy cells lacking normal expression of MHC-I molecules on the cell surface, due to an absence of an inhibitory signal that is received through inhibitory receptors that recognize and bind to ‘self’ MHC class I molecules. Thus, cells that express the normal level of host MHC class I molecules are protected from NK cells cytotoxicity (12). The missing-self hypothesis is supported by finding that transformed and viral infected cells usually tend to down-regulate MHC class I surface expression on its host (13), thus making them susceptible to NK cells attack (14). Transfecting MHC class I demonstrated an important validation of the “missing self” hypothesis- deficient target cells with MHC class I gene encoding D^d, K^b, and K^k. As a result, these cells become protected from lysis by NK cells (15). Further support for the “missing self” hypothesis demonstrated by typical rejection of allogeneic donors of MHC class I deficient bone marrow cells by wild-type mice was confirmed to be NK cell-mediated (16). The capability of an NK cell to recognize the lack of self-MHC class I expression on targets cells was linked to Ly49 inhibitory receptors. In 1989, Yokoyama and his colleagues identified an inhibitory receptor called Ly49 that is expressed on a subset of NK cells and that would be responsible for blocking NK cell activation. They purified and compared Ly49+ NK cells with Ly49- NK cells. They found a considerable number of target cells from H-2d and H-2k backgrounds, which were very susceptible to killing by the Ly49- NK cell subsets but were not killed by the Ly49+. Interestingly, mAb against Ly49 or the a1 and a2 domains of the H2Dd molecule was able to restore the killing of resistant target cells by Ly49+NK cells. Suggesting that interactions between MHC class I, and Ly49 inhibitory receptor will transmit an inhibitory signal that will turn off NK cell activation (17).

Human NK cells

Human NK cells constitute approximately 10% of the peripheral blood lymphocytes. They can be divided into two subsets based on their cell surface density of CD56. The majority of NK cells are CD3^–CD56^dim cells, which express high levels of CD16 (the FcgRIII), have greater cytolytic activity, play a key role in natural and Ab-mediated cell cytotoxicity, have an immunoregulatory function, and makeup around90% of circulating NK cells. On the other hand, 10% of NK cells are CD56^bright cells, which express low or no levels of CD16, and produce abundant cytokines (e.g., IFN-g). Therefore, the traditional phenotype of human circulating NK cells are CD3^–CD16^–CD56^dimor CD3^–CD16⁺CD56^bright (18, 19).

A balance between opposite signals delivered by the MHC class I–speciﬁc inhibitory receptors is responsible for the regulation of the effector function of NK cells. The inhibitory receptors are speciﬁc for HLA class I consist of three structurally distinct families: the killer cell Ig-like receptors (KIR), immunoglobulin-like transcripts (ILTs), and the killer cell lectin-like receptors (KLR) (20). Interestingly, KIR receptors were reported to be expressed on a considerable fraction of CD56^dim CD16⁺NK cells, where the CD56^brightCD16^–NK subset expresses uniformly CD94/NKG2A and lacks KIR receptors. Both NK cell subsets express the activating receptors NKG2D, which recognizes the MHC-class-I-related molecules MICA and MICB, as well as the natural cytotoxicity receptors (NCRs) NKp30 and NKp46 (21, 22).

The tissue distribution of these two major NK cell subsets depends on the distinct expression of chemokine receptors. CD56^dim CD16⁺cytotoxic NK cells can be attracted from blood to peripheral tissues by several chemokines released during inﬂammatory responses because of the expression of CXCR1 and CX3CR1. In contrast, CD56^brightCD16^– cytokine secreting NK cells express CD62L and CCR7, the receptor for CCL19 and CCL21 chemokines, which allow its migration from the bloodstream into lymph nodes (23).

Murine NK cells

Murine NK cells were initially characterized as a population of lymphocytes expressing the NK1.1 antigen. However, later on, NK1.1 was found to be only expressed in a few mouse strains, such as C57Bl/6 (B6), NZB, CE, FVB, and Swiss outbred mice, but not BALB/c, CBA, C3H, or 129 mice (24). Mouse NK cells are identiﬁed by the lack of CD3-TCR complex and by the expression of CD49b (DX5), CD11b, CD27, CD127, and NKp46 (25). Therefore, in NK1.1⁻ mouse strains NK cells are commonly identified using a monoclonal antibody (DX5) that recognizes CD49b, despite its expression on some activated T cells, platelets, and basophils (26). Interestingly, NKp46 were shown to be conserved between human, all strains of mice tested, and in three species of monkey. This made it the only unifying marker for NK cells across mammalian species, which would be defined as a true NK-specific marker however it was shown to be expressed by a very small subset of human and mouse T lymphocytes, including NKT cells (27, 28).

Many NK cells receptors that have been shown to activate or inhibit NK cell function, such as those belonging to theNKRP1, NKG2 and Ly49 families are encoded in the NK gene complex (NKC) that is located on chromosome 6 in mouse and chromosome 12p13.1 in human. Resistance or susceptibility of certain mouse strains to MCMV was linked to a mouse gene named Ly49H that is located in the NKC region. C57BL/6 mice, which express the Ly49H allele, are resistant to MCMV, whereas BALB/c mice, lacking Ly49H, are highly susceptible to MCMV infection (29). Many studies have shown that NKC appears to be a highly polymorphic region. Allelic variability of various NKC loci has been demonstrated in inbred mice and that involved in NK cell education (30).

NK cell receptors

General properties of NK cell receptors

NK cells express a variety of activating and inhibitory receptors including Ly49 or KIR, NKG2D, CD94–NKG2 heterodimers as well as natural cytotoxicity receptors. These receptors use opposing signaling motifs to inhibit or stimulate the activation of NK cells, with the negative signal mediated by MHC class I-speciﬁc inhibitory receptors being dominant over the activating signals. Inhibitory receptors allow NK cells to survey tissues for normal MHC class I expression and protect healthy cells from inappropriate NK cell-mediated killing (31).

Most NK cell inhibitory receptors such as inhibitory Ly49 family, KIR family, and CD49/NKG2A, signal through a common mechanism that is operating through immunoreceptor–tyrosine-based inhibitory motifs (ITIMs) which is present in their cytoplasmic tail. ITIMs motifs is consisting of Ile/Val/Leu/Ser-x-Tyr-x-x-Leu/Val (where ‘X’ represents any amino acid), the tyrosine residues in the ITIMs are critical elements for mediating inhibitory function. Ligation of these inhibitory receptors with its equivalent MHC-class I molecules leads to tyrosine phosphorylation of ITIMs motifs. Phosphorylated tyrosines in the ITIMs serve as docking sites that lead to recruitment of protein tyrosine phosphatase, Src homology region 2-containing protein tyrosine phosphatase (SHP)-1, to phosphorylated ITIMs. Recruited (SHP)-1 leads to disruption of activating responses (32).

Unlike the inhibitory receptors that contain an inhibitory motif in their cytoplasmic tail, most of activating NK cell receptors do not contain cytoplasmic domains capable of transducing signals. Instead, activating receptors signals is mediated by the association of the activating receptors with activating transmembrane adaptor proteins, such as DAP10 and DAP12, that contain an immunoreceptor tyrosine-based activation motif (ITAM), defined by the sequence (D/E)XXYXX(L/I)X6–8YXX(L/I). Engagement of these receptors leads to the phosphorylation of the ITAM tyrosine residues by Src family kinases. This leads to recruitment and activation of spleen tyrosine kinase, also known as Syk, ultimately leading to NK cells activation (33).

CD94/NKG2

Structurally CD94 and NKG2 belong to type II integral membrane glycoproteins that contain an extracellular C-type carbohydrate recognition domain. The CD94 protein were shown to be covalently assembled with distinct members of the NKG2, forming functionally distinct heterodimers. These receptors are expressed predominantly on NK cells and a subset of CD8+ T cells. This receptor varies in function as an inhibitor or activator depending on which type of NKG2 is expressed, activating (NKG2C, NKG2E/H) or inhibitory (NKG2A/B) isotypes (34). The ligand for CD94/NKG2 is a non-classical MHC I molecule, Qa1, in mouse and its homolog, HLAE, in human. HLA-E and Qa-1b molecules bind to peptides derived from the leader sequences of other MHC-I molecules (35).

NKG2A molecules present longer cytoplasmic tails containing ITIMs motifs in their cytoplasmic domains. Engagement of CD94/NKG2A with its ligand results in the inhibition of NK cell cytotoxicity (36). On the other hand, NKG2C and NKG2E have short intracellular regions and lack ITIM or ITAM motifs. These were found to be associated with the DAP12 for proper expression and initiation of activating signals (37). Although the activating CD94-NKG2C and CD94-NKG2E bind to the same ligand, their affinity for non-classical MHC-I molecule is much lower than the affinity of CD94-NKG2A (38). Interestingly, CD94-NKG2E plays an important role for NK Cell-Mediated Resistance to the Orthopoxvirus ectromelia virus (39). Moreover, NKG2C were also found to be able to recognize and kill of HIV-infected cells. During HIV infection the expression of the NKG2C receptor on NK cells as well as its ligand was shown to be unregulated, which could indicate its protective role during HIV infection (40).

NKG2D

NKG2D is a type II transmembrane protein that is a member of the C-type lectin family. NKG2D gene exists within the NK gene complex on human chromosome 12 and mouse chromosome 6. Although the NKG2D gene is located next to the other NKG2 genes in the NK gene complex, NKG2D displays only limited sequence homology to other NKG2 family members and it does not form heterodimers with CD94. NKG2D is expressed as a homodimer and signals through association with an adaptor protein, DAP10 in humans, and DAP10 and DAP12 in mice, which also help in stabilizing its surface expression. NKG2D is constitutively expressed on all NK, subsets of T cells, activated CD8+T and macrophages (41).

Several ligands have been identified for human NKG2D, including major histocompatibility complex class I chain-related molecules A and B (MICA and MICB) and UL-16 binding proteins (ULBP-1, -2, -3, -4, and -5), whereas mouse NKG2D bind to Rae-1α, Rae-1β, Rae-1γ, Rae-1δ, Rae-1ε, and histocompatibility antigen 60 (H60). Those self-molecules have been shown to be expressed in response to several stress conditions such as viral infections and DNA damage. Engagement of NKG2D with one of these self-molecules activates NK cell cytotoxicity and induces cytokine production (42).

In vivo and in vitro studies have demonstrated that mice were successfully able to eliminate tumor cells expressing NKG2D ligands. In human cancer patients, NKG2D ligands are constitutively expressed in multiple types of tumors, including AML (acute myeloid leukemia), ALL (acute lymphatic leukemia), CML (chronic myeloid leukemia), and CLL (chronic lymphatic leukemia) (42).

NKp46

One important family of activating receptors that are expressed on NK cells is the natural cytotoxicity receptors (NCRs) which include NKp30, NKp44 and NKp46. NKp46 is a type I transmembrane glycoprotein with 2 extracellular C2-type Ig-like domains and contains a charged amino acid in their transmembrane domain which associates with ITAM-bearing adaptor molecules CD3ζ and/or FcεRIγ. NKp46 has been shown to be expressed in both human as well as mice NK cells. Indeed, NKp46 is considered a major NK lysis receptor and plays a dominant role in the activation of NK cells against various targets furthermore is involved in the clearance of both tumor and virus-infected cells (43). Interestingly, Hemagglutinin molecules of influenza virus and the haemagglutinin–neuraminidase of parainfluenza virus were identified as the first specific NKp46 ligands (44). Although NKp46 receptors can recognize and kill tumor cells, however, the nature of these ligands is still unknown (45).

Mice lacking the NKp46 receptor fail to clear the influenza virus and do not survive the infection. A recent study has demonstrated that NKp46^high NK cells were more efficient at controlling HCV-infected hepatocytes than NKp46^lowNK cells (45).

Killer immunoglobulin-like receptors (KIRs)

KIRs are typed I transmembrane glycoproteins that are expressed predominately on NK cells and small subsets of T cells. KIRs are named based on their extracellular domain (2D and 3D), which reflects the number of immunoglobulin-like domains. Moreover, KIRs consist of both inhibitory and activating receptors, activating KIRs are characterized by their short cytoplasmic tail, whereas Inhibitory KIRs have a long cytoplasmic tail. The only exception is KIR2DL4, which is activating receptor with a long cytoplasmic tail (46)KIR gene family contains 15 genes and 2 pseudogenes with substantial allelic diversity of many of these genes that are closely linked on human chromosome 19q13.4 within the leukocyte receptor complex (LRC). Each KIR gene was shown to encode either an inhibitory or an activating KIR (47).

Studies of KIRs genotype demonstrated variations in the KIRs gene content depending on the individual. Based on the genetic diversity and allelic polymorphism of KIRs at the level of the locus, two main KIRs haplotypes can be distinguished: A and B. Generally, A haplotypes have encoded mostly for inhibitory KIRs (KIR2DL1, KIR2DL3, KIR3DL1, KIR3DL2, and KIR3DL3) and include KIR2DS4 and KIR2DL4 as the only activating KIRs, whereas B haplotypes are deﬁned by the presence of one or more of the following genes: KIR2DL5, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS5, and KIR3DS1, most of which are activating. Only three common genes (called framework) are shared by all haplotypes KIR3DL3, KIR2DL4, and KIR3DL2 (48-50).

Ligands for several KIRs have been defined, and every receptor appears to recognize a set of classical HLA class I molecules. The inhibitory KIRs contain ITIM motifs in their cytoplasmic domains and they interact with MHC class I molecules with various allelic specificity. For an instant, KIR2DL receptors were found to recognize predominantly the HLA-C alleles, whereas The KIR3DL2 receptors bind HLA-A3 and HLA-A11. On the other hand, activating receptors possess a lysine residue in their transmembrane domain allowing for association with DAP12, a molecule that possesses an immunoreceptor tyrosine-based activating- motif (ITAM). Excluding, KIR2DL4 that has a long cytoplasmic tail, was found to be associated with FcεR1γ, adaptor molecules containing the ITAM, to transduce an activating signal (51-53). Interestingly, some pairs of activating and inhibitory receptors, such as KIR2DS1 and KIR2DL1, recognize the same ligand (HLA-C), however activating KIR interact with HLA class-I molecules with lower affinity than their inhibitory counterparts (54).

Ly49 family

Ly49 family members are type II transmembrane glycoproteins with extensive homology to C-type lectin superfamily, structurally characterized as disulphide-linked homodimers. Ly49 genes are encoded in the NK gene complex on mouse chromosome 6. The Ly49 receptors are extremely polymorphic, with variations in gene number that exist in multiple allelic forms. Mapping experiments and complete sequencing of this locus of the Ly49 gene cluster using different inbred strain genomes (C56BL/6, 129/J, BALB/c, and NOD mice) showed distinct numbers of Ly49 genes in each mouse strain. For an instant, The C56BL/6 (B6 mice) Ly49 cluster is composed of two activating receptors (Ly49d, h), eight inhibitory receptors (Ly49q, e, f, i, g, j, c, a), and five pseudogenes that do not code for any functional proteins. In contrast, the 129-Ly49 cluster is composed of three activating receptors (Ly49r, u, p), nine inhibitory receptors (Ly49q1, e, v, ec2, s, t, i1, g, o), and seven pseudogenes (55). The majority of the Ly49 receptors are expressed by mature NK cells with an exception for Ly49E, Ly49B, and Ly49Q. Ly49E is expressed on fetal NK cells then disappears on mature NK cells. On the other hand, Ly49B and Ly49Q are not expressed completely by NK cells, although they are located on the same Ly49 gene complex (56).

Although mouse Ly49 receptor families are not structurally related to human KIRs, the Ly49 family members were demonstrated to be functionally equivalent to human KIRs. The ligands for Ly49 receptors have been demonstrated to be either major MHC-class I molecules or MHC-class I related molecules that are expressed by pathogens upon infection. Individual Ly49 receptors can recognize several, but not all, polymorphic MHC class I alleles. For example, Ly49A can recognize and bind to H2-D^d, H2-D^k, and H2-D^p, but not to H2-D^b, H2-L^d, or H2-K^b. and that makes Ly49A not functional in B6 mice, which express only two types of MHC-class I molecules (H2-K^b and H2-D^b) (57). Nevertheless, both Ly49C and Ly49I are extremely functional in B6 mice since they are able to recognize and bind to the H2-Kb molecule. If a target cell does not express the specific MHC I molecule that would be recognized by the available inhibitory Ly49 receptors on a given subset of NK cells will be lysis (58).

Inhibitory Ly49 receptors

The inhibitory Ly49s, such as Ly49A, Ly49C, Ly49I, and Ly49G, contain ITIMs in their cytoplasmic domains that become phosphorylated in response to receptor ligation with its MHC class I ligand on the target cell. This results in tyrosine phosphorylation of the ITIM, leading to recruitment of SHP-1 phosphatase, which results in dephosphorylation and deactivation of signaling proteins, the nucleotide exchange factor VAV1, involved in the NK activation cascade, thus blocking NK activation signals as consequence inhibit NK cells cytotoxic function (59).

Many tumors have been shown to express sufficient levels of self-MHC class I that in turn are recognized by Ly49 inhibitory receptors, thus making them able to escape lysis by NK cells. For example, In H2b strains of mice (B6 or 129 mice) the tumors bearing MHC H2b will grow smoothly without being killed by NK cells because of the strong interaction between the Ly49 inhibitory receptors (Ly49 C and I) and the tumor. However, blockade of Ly49C and I inhibitory receptors using F(ab’) 2 fragments of the 5E6 monoclonal antibody(mAb) resulted in increased cytotoxicity against these types of tumors and decreased tumor cell growth in vitro and in vivo (60).

Attempting to evade T-cell recognition, transformed and virally infected cells tend to down-regulated MHC class I from the surface of infected cells. This induces NK-mediated target cell lysis because of loss of inhibitory signals via self-MHC–recognizing receptors which induce NK cell cytotoxicity (61).

Activating Ly49 receptors

Activating Ly49, receptors lack ITIM sequences. Rather, their cytoplasmic domain is associated with ITAM-containing adaptor molecules, such as DAP12. Upon engagement of an activating Ly49 receptor, the ITAMs of the associated DAP12 become phosphorylated most likely by Src family kinases (including Lck, Fyn, Src, Yes, Lyn and Fgr), leading to the recruitment of protein tyrosine kinases ZAP-70 or Syk, which in turn initiates a cascade of signaling events leading to NK activation. It comes into sight that, when NK cell engages with healthy cells that express the normal level of MHC class I, inhibitory receptor signals are dominant over activating receptor signals by recruiting tyrosine phosphatases such as SHIP-1 to dephosphorylate appropriate kinases, preventing auto aggression and thus maintaining NK self-tolerance (62, 63).

NK cells from C57BL/6 mice express activating Ly49D receptors that are capable of recognizing and killing target cells that express MHC class I molecule, H-2Dd. Ly49D+NK cells in B6 mice play an important role in the rejection of bone marrow cells that were obtained from Balb/c mice, which express normally H-2D^d class I molecules. Additionally, Depletion of the Ly49D+NK subset resulted in increased engraftment of bone marrow cells in recipient mice (64). Interestingly, Ly49D was also shown to recognize MHC class I like molecule, Hm1-C4 that is normally expressed by Chinese hamster ovary (CHO) cells. Thus, make these cells to be extremely susceptible to lysis by Ly49D+ NK cells. Correspondingly, lysis of this target cell can be specifically inhibited using an antibody against Ly49D receptors to block the interaction between the receptors and its ligand (65, 66).

Several types of Ly49 activating receptors (Ly49H, Ly49P, Ly49I) were found to bind to viral MHC class I like molecules. Interestingly, both the Ly49H activation receptor (express in C57BL/6 (B6) mouse) and Ly49I inhibitory receptor (express in 129/J mouse) bind specifically to m157, and MCMV-encoded glycoprotein with an MHC class I-like homology. The binding of the Ly49H receptor to m157 makes B6 mice resistant to MCMV infection, whereas 129 mice are not. Moreover, Ly49h-deficient C57BL/6 mice were shown to be suitable for the MCMV infection. On the other hand, although BALB /c mice are susceptible to MCMV infection, BALB/c-Ly49H transgenic mice become resistant to the infection. That demonstrates the important role of activating Ly49 receptors during viral infection (67, 68).

Influenza A virus

General future and classification

The influenza viruses are classified as members of the Orthomyxoviridae family, which are defined as enveloped viruses with a segmented, negative single-stranded RNA (ssRNA) genome that contains 7–8 gene segments. Based on the antigenic character of influenza virus nucleoproteins, influenza viruses are divided into three types, influenza A, influenza B and influenza C viruses. Influenza A and B virus contain 8-gene segment whereas influenza C virus contains 7-gene segment. Structurally, unlike Influenza A and B which express two surface glycoproteins the hemagglutinin (HA) and neuraminidase (NA), the Influenza C virus expresses only a single glycoprotein, the haemagglutinin-esterase-fusion (HEF) protein, providing both of HA and NA functions (69, 70).

Influenza C viruses can cause only mild upper respiratory tract infections. However, influenza A and B viruses can cause human illness including upper and lower respiratory tract infection, and pneumonia. Influenza A can spread among both humans and animals including pigs, horses, mink, marine mammals, and birds, and are associated with the major human pandemics. Whares influenza B and C affects predominantly humans, however, it has been also isolated from seals and pigs, respectively (69, 70). Influenza A viruses are further classified based on genetic and antigenic differences in their HA and NA surface glycoproteins into many different subtypes. To date sixteen subtypes of HA (H1–H16) and 9 antigenic subtypes of NA (N1–N9) have been identified, all of which have been isolated from avian hosts. Theoretically, 144 possible different combinations of HA with NA protein could be found, over one hundred subtype combinations have been identified in birds so far (71, 72). Human influenza viruses of the subtypes H1N1 and H3N2 are the major cause of annual epidemics every year in the human population. Additionally, avian viruses H5N1, H7N7, H9N2, and H7N3 were also reported to infect humans (73).

Biology and life cycle of influenza A virus

The influenza A viruses are highly polymorphic and could be visualized as spherical or filamentous. Additionally, it is an enveloped virus with the outer layer composed of a plasma membrane obtained after its budding from an infected host (74). The influenza A virus genome enclose eight negative single-stranded RNA, that encode for HA, NA, viral matrix protein (M1), integral membrane protein (M2), nucleocapsid protein (NP), the RNA polymerase complex (PA, PB1, and PB2), and nonstructural proteins (NS1 and NS2) (Figure 1). Each genome segment is packaged in the virus in complex with the nucleoprotein (NP) and associated with the viral polymerase complex to form viral ribonucleoprotein complexes (vRNPs) (75). HA mediates influenza viral attachment and fusion to the target cell membrane by binding to sialic acids residues that are expressed on the target cell. Therefore, HA has an important role in determining host tropism. Human influenza virus has an HA receptor-binding speciﬁcity for sialic acid in a (2-6)-linkage [Neu5Ac (α2-6) Gal], whereas avian influenza virus higher speciﬁcity for a (2-3)-linkage [Neu5Ac (α2-3) Gal]. In parallel with these preferential binding properties, human airway epithelial cells were found to express mainly a (2-6)-linkage, and duck trachea and intestine contain mainly a (2-3)-linkage, moreover, In the pig trachea, epithelial cells contain both linkages which explain the capability of human and avian viruses to infect pigs (76, 77). Upon binding to sialic acids on the cell surface of the target cell, the virus is internalized into the endosome by receptor-mediated endocytosis. The lower PH of the endosomes activates the influenza M2 protein to pump in more protons (H+) into the vesicle, which acidifies the viral interior and facilitates the M1 dissociation from RNPs and release of the viral RNP segments into the cytoplasm. The RNPs are then imported into the nucleus, which is the major site for influenza virus transcription and replication. Viral RNA serves as a template for the synthesis of mRNA and cRNA. Newly synthesized HA and NA proteins are transported to the cell surface where they integrate into the cell membrane and initiate the budding event. Later on, the newly synthesized RNPs bind to M1 and that induces their export of the complex from the nucleus to the cytoplasm (70, 78-80). In the cytoplasm, the Interactions between M1 coupled with RNPs and the cytoplasmic domains of HA and NA, which serve as docking sites for M1, trigger the assembly of viral components at the lipid rafts and thus signals for exclusion of host proteins from the budding site. The last stage in the influenza A virus replication cycle is mediated by NA. Cleaving sialic acid residues from viral proteins, that preventing the HA-receptor interaction and aggregation of the new viruses. Consequently, allows the release of newly virions particles from the host cell surface to begin a new round of infection (70, 78-80).

Innate response to influenza virus infections

Both the innate and adaptive immune responses are responsible for host defenses against influenza infection. Although adaptive immune responses including T and B cells are important in clearance and prevention of influenza infection, however, it takes around 5 to 7 days before specific antigen-specific antibody and T cell traffic to the lung. thus during that time period, the innate immune cells including natural killer cells, alveolar macrophages, and dendritic cells (DC) play a critical role in host defense against virus infection by limiting influenza virus replication and enhancing the rapid development of adaptive responses (81). Invading pathogens, such as viruses or bacteria express several distinct ligands, known as pathogen-associated molecular patterns (PAMPs), which are essential for survival and pathogenicity such as Gram-negative outer membrane lipopolysaccharides (LPS), Genomic viral DNA or RNA. These molecular patterns are typically present on the pathogens’ surface or their nucleic acid. The mammalian innate immune system plays an important role in the rapid recognition and elimination of invading microbes through germline-encoded pattern recognition receptors (PRRs) that recognize the molecular signature PAMPs. PRRs can be cell-associated that are expressed intracellularly or extracellularly on the cell surface, such as TLR (Toll-like receptor) family. The stimulation of PRRs leads to the induction of several extracellular activation cascades such as complement pathways and various intracellular signaling pathways, leading to inﬂammatory responses that are essential for effective clearance of evading pathogens. Most of the innate immune cells express one or more of the PRRs such as macrophages, dendritic cells (DCs), mast cells, neutrophils, eosinophils, and NK cells (82-84).

Influenza A virus primarily infects lung epithelial cells and then spread to nearby epithelial cells and alveolar macrophages. Being a lytic virus, numerous influenza virus particles are released in the extracellular space and that exposes those influenza virus particles to innate PRRs, and by the infected, epithelial cells themselves. Infected lung epithelial cells were shown to detect the influenza virus replicative intermediate double-stranded RNA (dsRNA) using its Toll-like receptor 3 (TLR3), resulting in the production of Type 1 interferon-b (FN-b) (85, 86). Type I IFN (IFN-a and IFN-b) are a major component of the innate immune response that limits influenza viral infections and drives adaptive immune response to the site of infection by enhancing the presentation and recognition of influenza virus antigens. Moreover, IFNs stimulate the induction of several antiviral genes that interfere with influenza virus replication and thus contributing to cellular resistance to influenza virus infection. For example, IFN-induced expression of human MxA protein, which is capable of binding to the RNA polymerase subunit of the influenza virus and that prevent virus replication. Moreover, IFNs enhance significantly NK cell activity leading to NK cells proliferation and production of cytotoxic granules that kill the target cell. Moreover, IFNs enhance DCs differentiation and activation (87-89). Interestingly, DCs are also able to detect and recognize influenza virus single-stranded RNA (ssRNA) using its TLR7 and thus lead to robust induction of type 1 interferons (90).

In the resting state, alveolar macrophages negatively regulate NK cells activity by secreting inhibitory cytokines prostaglandins and transforming growth factor (TGF)-b). Studies have demonstrated that pulmonary NK cells from bronchoalveolar lavage (BAL) or from lung tissue were not able to lyse NK-sensitive target cells. However, incubation of pulmonary NK cells for 24h with IFN-I was enough to restore NK cell activity. Interestingly, NK cells from mice lacking IFN-I receptors were unable to kill MHC-class I deficient cell line (RMAS) in compression to WT NK cells after stimulation with IFN-I, however, both groups were able to recognize and kill another cell line that expresses a ligand for NK cell activating receptors, NKG2D. Thus, demonstrate the ability of NK cells to kill target cells that express stress ligand without previous activation. Moreover, Demonstrates the importance of type I IFNs as an early and critical regulator of NK cell activation and proliferation (91, 92).

Upon influenza virus infection, the infected macrophage produces a high amount of monocyte chemoattractants, particularly the CC chemokines (CCL2), which recruit large numbers of NK cells to the lung within the first few days of infection (93-95). Abundant secretion of IFN-I by activated and infected macrophage and DC augment NK cells cytotoxicity. Furthermore, direct interactions between influenza virus-infected- macrophage or DC with NK cells have strongly stimulated NK cytotoxicity and induction of IFN-g production, which has an important role in macrophages and DCs activation and Th1-type cell proliferation (96, 97).

Role of NK cells during influenza virus infection

Mice and hamsters depleted of NK cells showed increased morbidity and mortality during influenza virus infection, and that demonstrated the important roles of NK cells during influenza virus infection (98). NK cells are recruited to the lung two days after influenza virus infection and peak at day 5, whereas influenza virus peaks within 2 to 3 days after infection and declines significantly by day 5. The protective functions of NK cells during influenza virus infection were further specifically confirmed by the fact that NK cells activating receptor (NKp46) can recognize influenza hemagglutinins on virally infected target cells and this recognition is crucial for protecting mice against lethal doses of influenza virus infection. The binding of NKp46 activating receptor to the influenza virus hemagglutinins on infected cells triggers the NK cell to lyse the infected cell, consequently limiting viral infection and replication (44, 99, and 100). As seen earlier, influenza virus-infected monocytes and dendritic cells robustly enhance NK cells cytotoxicity by producing a high level of type I IFN, and by direct contact. A recent study has demonstrated that influenza virus-infected monocytes and dendritic cells express a high level of stress ligand UL16-binding protein (ULBP)1–3, which is recognized by NK cell activating receptor NKG2D, and that enhances cytolytic activity of NK cells toward influenza virus-infected cell and increased IFNγ production (97, 101). Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induces apoptosis of various tumor cells but not normal cells. Interestingly, influenza A virus infection was shown to induce TRAIL expression on lung NK cells. Interestingly, 4 days post-influenza virus infection TRAIL expression was detected only on NK cells but not on T cells. Blocking of this receptor results in increased virus titer significantly. Therefore, NK cells expressing TRAIL may play an important role in the immune response to influenza A virus infection (102). Additionally, NK cells are able to kill influenza virus-infected cells that are identified by a specific antibody (Ab) that binds to them. The protective function of M2-specific antibodies that recognize and bind to M2 protein expressed on the surface of influenza virus-infected cells depends mainly on the presence of NK cells to confer protection in vivo. NK cells mediate this protection via Antibody-Dependent Cellular Cytotoxicity (ADCC). NK cells express a receptor called CD16 that binds to the Fc portion of the Abs, this binding induces NK cell lytic function and kills the target cell (103, 104).

How influenza viruses can escape NK cell recognition?

In response to NK cell cytolytic potent function, the influenza virus has developed several evasion strategies to escape NK cells recognition. Interestingly, influenza viruses have developed strategy make them able to down-regulated NK cells activating receptors (NKp46) in vitro and in vivo. The study has demonstrated that, incubating influenza virus particles with NK cells resulted in significantly NKp46 down-regulation from the cell surface of NK cells (105). In agreement with this finding, incubation of fresh or IL-2-activated NK cells with influenza virions or hemagglutinin resulted in a significant inhibition in NK cell cytotoxicity toward influenza virus-infected macrophage (106).

Additionally, the influenza virus was found to be able to bind directly to sialic acids residue that is expressed normally on the cell surface of NK cells. As a result, NK cells become extremely susceptible to influenza virus infection, which induces striking NK cell apoptosis. Influenza virus uses clathrin-dependent endocytosis as an entry pathway. Although some influenza viral components have been synthesized, however, no infective virus was produced, abortive infection (85). In vivo infection of lung NK cells by influenza, A virus was also confirmed. NK cells express both sialic acids Alpha-2, 3 and Alpha-2, 6 linkages that facilitate influenza viral binding and entry. Additionally, this infection resulted in significantly lower cytotoxicity of influenza-infected NK cells than uninfected-NK cells against NK cells-sensitive target cells, YAC-1 and RMA/S (107).

As well known, NK cell activity is regulated by a variety of both activating, such as NKp46, and inhibitory receptors, such as inhibitory Ly49 family in mice and KIR in humans. Upon engagement of both activating and inhibitory receptors to a target cell, the outcome is determined by the net balance of signals, which determines whether the NK cell becomes activated to kill the target cell or not (108). Surprisingly, influenza virus infection was shown to induce reorganization and accumulation of MHC class I molecules in the lipid raft microdomains of the infected cell. Leading to increases binding of the NK cell inhibitory receptors, KIR2DL1, therefore NK cell cytotoxicity was significantly decreased (109, 110). Other observations demonstrated that influenza virus infection enhances greatly up-regulation of MHC class I molecules on infected human alveolar epithelial cells, A549 cells, and that would inhibit NK cell cytotoxicity through (111). To our knowledge, there is presently no in vivo infection model to demonstrate that, the influenza virus induces up-regulation of MHC class I on infected epithelial cells. If so, would that inhibit NK cells from targeting infected cells?

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