Natural killer cells in autoimmunity : clues or tools ?

Natural killer (NK) cells are large granular lymphocytes involved in clearance of damaged cells such as neoplastic and virus infected cells. Despite usual classification, NK cells are now grouped among the so called immune lymphoid cells (ILCs) which play fundamental roles in innate immunity and also in tissue remodeling. As other ILCs, NK cells are characterized by a wide spectrum of functions and participate in various phases of immune system activation or suppression by producing large amounts of soluble mediators. They are classically involved in cytotoxic responses even if they can regulate other immune cells by cell-cell interactions and can also generate memory subsets linking them to adaptive immunity. In spite of the intrinsic difficulties to investigate NK cells role in autoimmune diseases, data increasingly suggests that these lymphocytes could operate in self-tolerance breakdown but also in the control of autoimmune reactions playing a double role. In fact, NK cells can directly down regulate autoimmune responses by acting on immune cells or by diminishing the exposition of auto-antigens even if, they can enhance a pathological state of autoimmunity stimulating both natural and adaptive immune compartments. This review is focused on recent findings on NK cell biology, classification, activation, trafficking and organ distribution but also on interactions with other immune cells. It will explore their tasks in autoimmunity ranging from connective tissue diseases to allergic conditions with particular attention on the most recent studies. It will discuss NK cells putative role in breakdown or selftolerance maintenance and the possibility to manipulate them for treatment purposes.


Introduction
Natural killer (NK) cells are a class of lymphocytes characterized by a wide spectrum of functions ranging from effector responses to the ability to influence various steps of immune system activation by producing soluble mediators.NK cells were discovered in the mid-seventies as a subset of lymphocyte [1] with the ability to lyse tumor cells in vitro.They represent approximately 5-15% of human peripheral blood lymphocytes and are recognized as one of the mainstay of the innate immunity compartment involved in defense against tumor cells and virus infected cells.However, there is increasing evidence that NK cells have not only the ability to kill modified or infected cells but also to enhance adaptive immunity processes by producing large amounts of soluble mediators under specific signals [2] .Recent studies have demonstrated how even NK cells have the ability to develop memory REVIEW functions, in fact they respond vigorously to already known signals [3] playing critical roles in second response against pathogen noxa.These findings lead to define these cells as more than innate immunity cells, complicating our understanding of their role in a healthy immune system but also in pathological conditions.With concern to the pleiotropic range of actions, NK cells are now under investigation as one of the primary actors involved in the breakdown of immune tolerance.Growing evidence shows that these lymphocytes not only can induce autoimmune mechanisms but can also regulate and suppress selfreactive processes with the interaction of both natural and adaptive immunity, indeed, recent studies have highlighted their ambivalent regulatory properties [4] .At the moment, these two different features are being studied by researchers because it appears of capital importance to known how, when and where NK cells perform the one or the other action.Disclosing NK cells pivotal functions in autoimmunity lead us to understand pieces of the more complex puzzle of self-reactive processes and can finally improve our capability to manipulate these cells for treatment purposes.The most recent findings about the NK cells biology and their role in autoimmune diseases are reviewed below.

Definition and classification
NK cells are widely studied lymphoid cells defined as large granular lymphocytes capable of killing a target cell without any specific priming even if with a complex activation procedure.At the time of their discovery it was immediately clear that NK cells' activation was not regulated by molecular targets expressed with major histocompatibility complex (MHC) as in other lymphocytes, but only by the presence or the absence of MHC class I (MHC-I) molecules on cells.For these reasons, NK cells have always been defined as a component of innate immunity.Over the years, NK cells have been well molecularly defined discovering several adaptive cell behaviors, different subtypes and even other structurally related lymphoid cells.These new lymphoid populations are defined as innate lymphoid cells (ILCs) and their importance is growing because they seem to play major roles in natural immunity and in tissue remodeling.ILCs are defined by three features: I) their lymphoid morphology; II) a lack of myeloid cell and dendritic cell (DC) phenotypical markers; III) the absence of recombination activating gene (RAG)-dependent rearranged antigen receptors [5] .All ILCs require the transcriptional repressor inhibitor of DNA binding 2 (ID2) and the common γ-chain of the interleukin-2 receptor (IL-2Rγc) for development [5,6] even if there are some differences between ILCs populations, in fact, although IL-15 is crucial for NK development, it is not necessary for the maturation of other ILCs subtypes [7] .Several ILCs populations have been discovered and maturation differences identified.For these reasons, in recent time, a new classification has been proposed based on cytokines spectrum production and development process [5] which describes three ILCs groups (Table 1).NK cells are classified in Group 1 ILCs for their ability to produce large amounts of INF-γ but not type 2 cytokines (IL-4 and IL-13), IL-17 or IL-22.

Development, subsets and distribution
The development of NK cells is still not completely understood, however it is known that the process starts in the liver during fetal life and then after birth occurs in bonemarrow.Subsequently the description of a common lymphoid progenitor (CLP) a NK cells precursor (NKP) was defined and called CD122 + NK1.1 -Lin -cell [8] .This population of cells has the ability to maintain the potential to evolve towards a T cell or NK cell in vitro and in vivo [9] .Studying NKP in mice, it has recently been possible to identify a new precursor which could help to better define NK and ILCs developmental process in humans [10] .The NKP precursor is characterized by the expression of high levels ID2 and the IL-7 receptor α-chain (CD127) but lack most of NK cell markers [10] .Probably, NKP precursor is the key to understanding developmental processes in human ILCs but even if tests on mice could advance our knowledge, we are still far from a complete definition in humans.At the present it is not possible to detect the precise checkpoint from which this common progenitor is irreversibly engaged towards NK cells or others ILCs during the maturation process.NK cells late developmental process is well characterized and leads to define human mature NK as CD56 + CD3 -cells, distinguishing from a mixed population of NK-like T cells CD56 + CD3 + that have up-regulated several NK cell markers [11] .In humans it is possible to identify two major NK subsets which differentiate from each other, for functions, distribution in tissues and for superficial markers.These are called CD56 bright and CD56 dim on the basis of the amount of CD56 expression [12,13] .CD56 bright NK cells are well represented in A: Protective role.NK cells could protect the organism from autoimmune reactions acting on different levels, in fact, they can control auto-antigens exposition clearing damaged cells such as viral infected cells, controlling thus some of the environmental factors.Furthermore, NK cells seem to be able to kill immature DCs and auto-antigens presenting DCs but also auto-reactive T cell clones.It has been demonstrated that NK cells can eliminate hyper-activated macrophages monitoring tissue injuries and antigens presentation but can also enhance T regulators lymphocytes activities which down regulate auto-reactive T lymphocytes.By producing cytokines as IL-5, IL-10 or TGF-β, NK cells are able to directly modulate adaptive immune response reducing CD4 + T cell interaction with B cell which probably decreases the auto-antibodies production.B: Promoting role.The hypothesis of promoting role on autoimmune diseases demonstrates how NK cells may lose self-tolerance, inducing or participating in maintenance of autoimmunity.This process could be based on an altered expression of inhibitory KIRs which activate NK cell natural cytotoxic potential leading to direct tissue damage with a subsequent abnormal exposition to auto-antigens.Moreover, NK cells could be activated by healthy cells covered by auto-antibodies causing injuries and perpetrating autoimmunity.NK can also increase adaptive autoimmune process inducing DCs maturation in inflamed tissues, but also enhancing antibodies production, stimulating auto-reactive CD4 + T cells, promoting differentiation of naïve CD4 + T cells and increasing CD8 + T cells activation.NK cells are able to enhance macrophages activities such as antigen presenting cells role and cytokines.
secondary lymphoid tissues, they make up 5-15% of total mature NK cells and are the more immature compared to CD56 dim even if they exert a powerful capacity to product large amounts of soluble mediators they are not well able to build cytolitic reactions [11] and express low levels of CD16/FcγRIIIa.On the contrary, CD56 dim constitute approximately 90% of NK cells, they are located in peripheral blood and mediate predominantly cytotoxic reactions with a lesser capability to produce cytokines in respects to the other subset but expressing high level of perforin, granzyme B and CD16/FcγRIIIa [11,14,15] .Increasing evidence depict CD56 bright NK cells as the precursor of CD56 dim NK cells [16][17][18][19] .According to this hypothesis a transitional cell stage between bright and dim NK subset has been identified in peripheral blood [20] and subsequent studies have further proven the process of maturation from CD56 bright to CD56 dim described above [21- 23] .Telomeres analyses have discovered shorter sequences in CD56 dim in accordance with a later appearance in the human body of this subset [24,25] .Albeit, there is not sufficient data to define NK cell tissue distribution, it seems that NK cells are present not only in peripheral blood and lymphoid tissue but also in skin [26] , joint [27] and other internal organs like gut [28] , liver [29] , lung [30] and kidney [31] .It has been discovered that NK cells are present even in the uterus during pregnancy where they play an important role promoting angiogenesis and tolerance towards cytotrophoblast during placentation [32][33][34] .

Biological functions and cell-cell interactions
NK cells most known actions are the killing of infected or tumorigenic target cells and by secreting inflammatory cytokines like interferon-γ (INF-γ) which coordinate but also activate other cell types.These responses are based on a complex interaction between surface molecules expressed by NK cell and the potential target [35] .The first explanation of NK cell activity was defined as the "missing self" theory and derives from experiments in mouse models [36,37] .This theory clarifies how NK cells are intrinsically ready to kill their targets and how they are able to eliminate any cell that lacks MHC-I molecules [38] (Figure 1A).However, such great potential can be dangerous if it is not finely controlled, in fact, further studies have demonstrated how NK cells natural cytotoxicity is under the influence of activating or inhibiting signals which results from stimulation of their widespread surface receptors.During recent years, a large spectrum of receptors has been discovered to participate in NK cells regulation, some of these mediate antibody dependent cytotoxicity (ADCC) and others are activated only by ligands produced during inflammation which ultimately bind NK cells to autoimmunity [39] (Figure 1A).In humans, the predominant NK cell receptors are killer-cell immunoglobulin-like receptors (KIRs), a widespread class that recognize specific human leukocyte antigen (HLA) class I allotypes and can mediate both activating and inhibiting signals (Figure 1A).Description of NK's receptors is a purpose which goes beyond this article but these are well reviewed elsewhere [40,41] .NK cells activation is mediated by the constitution of one or more immunological synapses [42] with their targets.These connections lead not only to patrol several cells at the same time but also to simultaneously inhibit or activate the same NK cell on the different sides of its cytoplasmic membrane [42,43] (Figure 1A and B).Among other things, NK cells perform many tasks by producing a large array of cytokines both proinflammatory, like tumor necrosis factor-α (TNF-α), or immunosuppressive as interleukin-10 (IL-10).Increasing evidence demonstrates how NK cells have the potential to directly interact and regulate several immune cell types at different stages of the immune response, indeed, by producing a great spectrum of chemockines [44] , NK cells can influence the activity of DC, macrophages and neutrophils [45] but also regulate positively [46,47] or negatively [48] T and B cell functions.This evidence produces proof to affirm that NK cells are a pivotal link between innate and adaptive immunity.NK cells have to be primed by several factors to achieve their full regulatory potential, in fact IL-15 presented by DC [7] or macrophages [49] , IL-12 [50] or IL-18 [51] are crucial steps towards a complete maturation.Even CD8 + cytotoxic T cells can be influenced by the NK cells ability to promote antigen presentation through cell debris [46] or to decrease T cell activity by reducing the antigenic load [47] (Figure 1C).In addition, recent findings on mice have demonstrated the presence of mature long-lived NK cells that can produce a powerful recall response upon cytokine stimulation [52] .These cells behavior is quite similar to what memory lymphocytes exert during a so called second response of adaptive immunity where mature cells promptly react against an "already known" antigen.Ongoing studies are now focused to demonstrate this memory NK cells subset even in humans.Taken all together these data suggest how NK cells are strictly involved not only in primary response but also in modulating reactions of other cellular populations leading to review our common concepts on their natural immunity (Figure 1C).

NK cells tolerance and autoimmunity
Accumulating data demonstrate that NK cells are crucial not only in responses against virus and tumor but also in regulating effector reactions through cytokine secretions and cell-to-cell contact.These features can be used to control and prevent the attack of the immune system against healthy cells.However the most recent evidence appears controversial because it shows that NK cells could participate or suppress in autoimmune reactions.The explanation of these data may be related to the presence of different and not well defined NK cell subsets which could participate in autoimmune processes playing different and even opposite roles.Studying NK cells in autoimmune diseases results of difficult realization because it should be important to define their roles in the first phases of illness but this approach is no feasible due to the impossibility to detect autoimmune pathologies from the beginning.Even if studies in NK cells in autoimmunity are not so numerous, it has been possible to detect several NK cell changes both qualitative and quantitative in peripheral blood and tissues.The most frequent finding is a reduction in the number of circulating NK cells [53][54][55][56] and a parallel decrease in cytotoxicity [57] .These data can be interpreted as a consequence of natural chronic NK cells activation and apoptosis due to autoimmune process or drug treatments.The complexity to interpret NK cells data in autoimmune diseases is well explained by what occurs during varicella infection.It was reported that severe varicella was associated to an important deficiency of NK cells but was discovered that this finding is transiently caused by infections and not vice versa [58] .Important data that bind NK cells to autoimmunity derives from a rare condition called NK lymphoproliferative disease of granular lymphocytes (LDGL) [59,60] .In this pathology there is a chronic excess of NK cells which are normal from a cytological point of view but present changes in their surface receptors leading to a dysregulation of NK cells actions which produce tissue damages even if the molecular mechanisms [61][62][63] are not known.

Genetics
As a mainstay of tolerance breakdown, genetic susceptibility has been extensively studied in humans to ascertain if there are any predisposing elements.Studies have principally focused their attention on KIRs polymorphisms and their coupling with HLA class I ligands because defects and variations of KIR/HLA genotypes could impair NK cell activation and self-tolerance [64] .Some evidence indicates how there is an increased risk of autoimmune diseases if the subjects express a definite set of KIR/HLA, in fact, KIR2DS2 is related with systemic sclerosis, rheumatoid vasculitis, type I diabetes (T1D), and systemic lupus erythematosus (SLE) [65,66] .Other positive associations have been detected between activating receptor KIR3DS1 and the development of ankylosing spondylitis (AS) [67] or KIR2DL5 and KIR3DS1 genes with multiple sclerosis (MS) [68] .In psoriatic arthritis, the 2DS1/2DS2 genotype and HLA-Cw homozygosity were both associated to susceptibility to developing of the disease [69,70] .In addition several studies have shown clinical association of autoimmune diseases with FcγRIIIa, in fact some polymorphisms can influence human IgG binding but also ADCC function as in SLE nephritis [71] .Studies on NKG2D, one of the most widely investigated NK cell receptors, and MICA have demonstrated how their polymorphisms are linked with T1D, psoriasis and Behcet's disease [72,73] .

Tolerance and NK cells regulatory roles
Increasing data demonstrates a high presence of NK cells in some tolerant organs such as liver, lung, gut and uterus suggesting NK cells play a pivotal role in induction and maintenance of tolerance versus self and even foreign antigens [74] .Focusing the attention on the liver, it is well known that this organ hosts a numerous population of lymphocytes and among these a great part is constituted by an immature phenotype of resident NK cells [75,76] .These peculiar NK cells seem to actively induce tolerance against the copious antigens derived from gut digestive processes and maintain their immature status only in liver microenvironment [77] .In lung and the intestine, NK cells have strong relationships with other ILCs at mucous level where it seems to be important to mediate and reciprocally regulate human microbiota preventing inflammation [78] .All these findings suggest there is an educational process by which NK cells acquire different capacities to sense not only self but also bad and good non self-antigens.In fact, there are examples of MHC-deficient individuals with a normal developed population of NK cells that are not selfreactive [79,80] , finding that cannot be explained by "missing self" theory.The educational process, otherwise known as licensing, is finely tuned by several molecular interactions.Different signals, both activating and inhibiting produce cellular metabolic changes which results in production of mature cells.These licensed NK cells acquire effector function and exhibit greater responsiveness towards activating receptors but also they are tolerant with the engagement of inhibitory receptors by normal cells.Licensing is ruled by the strength and quality of cellular signals.Interestingly, it is clear that this process is not based on an on-off switch [81] and more important, it shows a reversible behavior which leads to NK cells adapting to environment changes [80] .Moreover NK cells can influence maturation and activity of other NK cells not only by inhibitory signals but also by stimulating activating KIRs that interact with specific and co-expressed ligand MHC-I molecules tuning down NK cell responsiveness [82] .Other evidence demonstrates the importance of activating signals in acquisition of a balanced tolerance, in fact a NK cell chronically exposed to activating stimuli, like natural killer-group 2, member D (NKG2D) ligands, becomes non-reactive versus NKG2Dligands expressing cells [83,84] .Albeit this is still not well defined, NK licensing is a fine tuned mechanism which plays an important role in the regulation of NK cells and indirectly of other immune cells.In fact, it is possible that during inflammation or viral infections, in a genetically predisposed subject, some steps necessary to develop tolerance could be bypassed leading to ignition of autoimmunity.

NK cells in autoimmunity
The role of NK cells in rupture of tolerance are widely debated because accumulating data indicate both protective and promoting actions in pathogenesis of autoimmune diseases.These findings are difficult to understand together, however it must to be highlighted that NK cells are a pleiotropic cell population which can change their "common" way to proceed in agreement with environmental stimuli.In fact, these lymphocytes can perform different or opposite tasks depending on their involvement in physiological or pathological process as well as from the organ where they are located.Moreover, several studies differ in methods of manipulating NK cells, experimental procedures or mouse models used thus producing data that are difficult to compare reciprocally.Others important variables are represented not only by the complex relations taken with local ILCs which are diverse for any organ and mucous membrane but also for the different stages of diseases that sometimes result in NK cells producing an opposite spectrum of cytokines [85] .For these reasons it is hardly possible to define the first key events that alter NK cells bringing them towards autoimmunity and so assign a precise place in selfreactions, but we can describe the successive dynamic equilibrium which can be involved in maintenance or rupture of tolerance.

The protective role
As part of natural immunity, NK cells participate in the early phases of immune defense.For this reason, NK cells play important roles not only acting against pathogen noxa but also enhancing and stimulating other immune cells to react.NK cells perform regulatory activities not only by cell-cell contact but also by secreting cytokines and interacting in particular with DCs influencing and monitoring subsequent innate and humoral events.The results of this relationship are of great importance because NK cells can mediate not only by killing immature monocyte derived DCs but can also manage the DCs maturation [86] .The NK cells monitor on DCs activities has been interpreted as a sort of control system that could allow a major number of mature and balanced DCs to migrate to lymph nodes.An impairment of the above explanation could be responsible for an unrestrained reaction even against self-antigens.Another means by which NK cell down regulate DCs activation is represented by production of IL-10, one of the main anti-inflammatory cytokines [87] .IL-10 can inhibit DCs activities reducing antigens presentation and subsequent T cells proliferation [88] leading to control immune and autoimmune reactions (Figure 2A).Moreover, NK cells down-regulate macrophages activity through direct killing or soluble mediators, in fact, cytolysis of activated macrophages can be triggered by NKG2D receptors [89] .These tasks highlight NK cells abilities to regulate and suppress auto reactive molecular loops and self-activated cells indicating proof of their protective role against breakdown of tolerance (Figure 2A).

The promoting role
The failure of NK cells self-tolerance can be derived from the removal of inhibitory signals but also from the excessive stimulation of activating receptors.NK cells can influence autoimmune reactions directly expressing their cytotoxicity, but also by secreting cytokines and via cell signaling.NK cells can promote autoimmunity enhancing DC survival, maturation and cytokines production but also priming CD8 + T cells and improving CD4 + T cells Th1 subset by producing INF-γ [90][91][92] .Moreover, it has been described as a direct tissue damaging by NK cells through their cytotoxic activity which lead to release selfantigens [93,94] (Figure 2B).Some examples of NK cells detrimental role are represented by kidney injuries in systemic lupus erythematosus (SLE) [95] , cytotoxicity against neurons and other nervous system cells in multiple sclerosis (MS) [65] , rheumatoid arthritis (RA) synovial inflammation where there is a pro-inflammatory NK cell subset that trigger monocyte differentiation to DCs amplifying inflammatory responses [93] .Recent studies show other human conditions where NK cells could play significant roles triggering the start to self-reaction, such as psoriasis, alopecia areata and pemphigus vulgaris [96] .

Systemic lupus erythematosus (SLE)
SLE is an autoimmune disease that primarily affects young women.From a clinical point of view, it is characterized by a chronic and indolent course with periodical flare-ups.It can affect different organs which progressively lose their functions under the constant injury of the autoimmune reaction [97] .SLE is the prototype of autoimmune diseases where the state of chronic non-tissuespecific inflammation lead to organ damage and a polyclonal B-cell activation determines the production of auto-antibodies against several self-antigens such as double stranded DNA [97] .In recent years, growing evidence has focused attention on the role of innate immunity as the principal trigger for the adaptive response.Indeed, findings suggest the role of neutrophils NET [98] and the defective control of apoptosis [99] as the main source of nuclear autoantigens exposition.Moreover, the association of NK cell impaired functions to SLE has been well demonstrated by the presence of predisposing KIRs expression, such as KIR2DS2 [66] , and FcγRIIIa polymorphisms [66,71] .Other results found in SLE patients are represented by a NK cells cytotoxic deficiency expressed by both lower perforins level [57] and a variable number reduction [56] .In fact, NK cells number alterations is related to some clinical manifestations such as nephritis and thrombocytopenia during flare-up of the disease [57] .Taking all this into consideration, these data are difficult to correlate in such a complex disease; in fact, these alterations could be due to a state of chronic inflammation or, on the contrary, could be the start of autoimmunity.Recent theories suggest the presence of impairment in both bone marrow production and peripheral organs maturation which leads to NK cell function alterations.However many questions remain unanswered and only further studies will be able to clarify the real roles of NK cell in SLE.

Rheumatoid arthritis and spondyloarthorpathies
Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovial inflammation which leads to cartilage and bone erosion resulting in joint destruction.NK cells activity in RA has been widely investigated with controversial results, in fact, some authors have reported a normal NK cell number while on the contrary recent studies suggest a reduced number and an impaired NK function [56,57] .The greater part of NK cells, in synovial membrane, is represented by CD56 bright subset which can be induced to produce large amounts of proinflammatory cytokines [14,100] by Th1 polarized reaction.In this molecular milieu, NK cells can directly interact and manage other immune cells leading to stimulation of B and T cells [100] .Moreover, the ability of NK cells to induce monocytes differentiation towards DCs [101] has been demonstrated, which, ultimately, could generate a selfsustained loop where new generated DCs augment NK cell activities.In accordance with the explanations in the paragraph on biology, NK cells seem to be involved in the regulation of joint inflammatory processes acting both by enhancing DCs secretion of pro-inflammatory cytokines and on the other hand suppressing DC maturations [102,103] .Considering HLA predisposition to develop RA, it has been possible to demonstrate how the activating receptor KIR2DS2 was positively associated in developing RA and RA related vasculitis in Taiwanese patients [104] .Spondyloarthopathies (SpA) are a group of chronic inflammatory diseases characterized by similar even if pleiotropic clinical manifestations which share a common genetic background particularly represented by HLA-B27 antigen.NK cell roles have to be clarified in spite of some recent evidence demonstrating that specific KIRs patterns expression are directly related to disease susceptibility.Moreover, NK cells seem to play relevant pathogenetic functions through stimulation of different activating or inhibiting KIRs [69,105] .One of the major contributions to the development of SpA and ankylosing spondylitis [105] , is ascribed to KIR3DL1 and KIR3DS1 alleles, which are positively related to diseases, whereas KIR2DS1 and KIR2DS2 have a protection role only if their respectively ligands are not lost [69] .These data suggest how the persistence and, probably, the breakdown of tolerance can be subscribed to NK cells activity, albeit, further studies are necessary to understand the real contribution of innate immune cells subsets in RA and SpA pathogenesis.

Autoimmune liver diseases
The liver is an organ with a complex structural parenchyma that harbors an unusually high number of immune cells.About 30-50% of total liver lymphocytes are represented by NK cells which are involved in control of immune tolerance versus the great amount of foreign antigens derived from gut digestion.In that unique microenvironment, a large number of cell populations interact producing a wide spectrum of cytokines.Disclosing the pathways involved in breakdown of tolerance in liver environment is complicated but some researches have focused their attention on NK cells discovering interesting findings in three principal autoimmune liver diseases: primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC) and autoimmune hepatitis (AIH).
PBC is is a chronic cholestatic liver autoimmune disorder.It is now clear how immune dysregulation, in particular adaptive immunity, is the foundation of liver injuries in this pathology, in fact, expansion of T and B lymphocytes with generation of anti-mitochondrial autoantibodies is a mainstay in biliary duct damages.However, growing evidence has demonstrated how innate immunity and NK cells contribute in PBC pathogenesis [106] .Studies show an increased number of NK cells in peripheral blood compared to healthy donors [107] , furthermore, these lymphocytes express high levels of perforin and express a greater cytotoxic activity against cholangiocytes [108] .Further proof is represented by the secretion of CX3CL1 and CXCL3 chemokines on biliary ducts which are responsible in recruiting NK cells in the liver and seem to mediate biliary ducts damage [107] .
PSC is a largely unknown pathology defined by sclerosing lesions of biliary ducts which lead to strictures and obstructions.PSC is characteristically associated to inflammatory bowel diseases (IBD) and recently a new category related to IgG4 diseases has been discovered.Numerous NK cells alterations have been described in PSC patients.Studies highlight a reduction in both NK cells number [109] and cytolitic activity [110] , however, recently it has been possible to identify an increased cholangiocytes destruction mediated by TRAIL-dependent mechanisms [111] which prove the important alterations of NK cells biology in PSC liver.
Little is known about NK cell actions in AIH.Most researches have focused their attention on tissue damage displayed by CD4 + and CD8 + T lymphocytes [112] , nevertheless, NK cells and macrophages participate actively in hepatic injuries not only with an effector role but also regulating other immune cellular populations [112] .

Multiple sclerosis (MS) and central nervous system (CNS) degenerative diseases
MS is a neuroinflammatory autoimmune disease which affects young adults causing widespread and progressive disabilities.It is characterized by a variable demyelination of white matter due to brain infiltration of immune cells which determinates clinical phases of inflammatory activity alternated by periods of remission.NK cells role has been widely investigated, studies demonstrate a reduction in NK cell numbers [113] as well as cell cytotoxic activities during reactivation of disease that are restored when MS is clinically dormant [114] .During pregnancy, an increased peripheral blood NK cell number has been discovered directly related to clinical remission [115] .Moreover, the use of the monoclonal antibody daclizumab seems to restore NK cell numbers, especially CD56 bright subset, in accordance with reduction of disease flare [116] .All together, these findings highlight a putative regulator role for NK cells in MS even if further studies are needed to prove eventual therapeutic applications.
CNS degenerative diseases are principally represented by Alzheimer (AD) and Parkinson (PD) disorders that are characterized by an age-related neuronal loss in specific and different brain areas.Some theories ascribe these pathological situations to an immune dysregulation [117] even if studies which focus on natural immunity have highlighted a normal NK cells count [118] in AD patients.However, an association has recently been described between an increased NK cells cytotoxic activity and the expression of 5-HT2c receptor for serotonin [119] .Instead, PD patients show two different situations: in the first condition NK cell cytotoxicity is diminished and related to an increased expression of inhibitory receptor NKG2A [120] , but the second demonstrates an increased cytotoxic activity and inflammatory cytokines production which are directly related to PD severity [121] .

Allergic diseases
Allergic diseases are one of the most growing pathologies in developed countries.At the present time, it has been demonstrated that there is an increasing incidence especially in early age.Moreover, these early conditions are aggravated by an insufficient pharmacological response which lessens their clinical control.Atopic diseases such as food allergy, allergic rhinitis, asthma and anaphylaxis have significant associated morbidity along with large health care expenditures.Allergy is a pathological condition characterized by an inappropriate immunologic reaction to harmless antigens principally driven by the so called type 2 helper (Th2) responses.It is now well known that the imbalance of Th1/Th2 cell polarization toward the last Th2 subset plays the major role in both initiation and maintenance of the allergic processes by producing large amounts of type 2 cytokine like IL-4, IL-5 and IL-13 [122- 124] .Albeit NK cells activities in atopic conditions are poorly described, recent studies highlight their role in the ignition of pathological reaction [125,126] .Among allergic diseases, it is possible to identify some of the major conditions studied such as atopic dermatitis (AD) and rhinitis which are both risk factors for asthma.In patients with allergic rhinitis, NK cell numbers were found to be significantly reduced even if their cytotoxic activity and Th2 cytokines expression were higher compared to healthy controls [127] .Moreover, increasing studies demonstrate how the modifications of cytokine milieu towards Th2 could impair NK cell-DCs interactions leading to the disruption of NK cells regulatory roles on other immune cells.Another example is AD, a chronic, pruritic and inflammatory skin condition that is associated with epidermal barrier dysfunction.AD is the most common skin disease in childhood [128] and is characterized by inflammatory cell skin infiltration leading to disruption of the skins normal structure causing both social and physical disabilities.It is well known that both adaptive and innate immunity are involved in AD pathogenesis [129,130] and, in particular, the most recent studies focus on the NK cells ability to regulate immunoglobulin isotype switch as an important regulatory pathway.It has been discovered that with the selective suppression of IFN-γ secretion the NK cells regulatory effect on Th2 cell populations were decreased and immunoglobulins type E production is directly augmented [131] .All the findings reported above indicate the direct role of NK cells in the control of Th2driven inflammation [132] and finally on allergic responses, even if, considering the new ILC populations, it is necessary to study and clarify NK cell roles revising all interactions and pathways in healthy and pathological stata.

Microbiota and NK cells therapy
Growing studies are focusing attention on the ability of commensal bacteria to influence human being health status modifying both innate and adaptive immunity.Indeed, every surface of our body is colonized by thousands of microorganisms which interact with human being cells.These relations are finely tuned and generate a complex dynamic equilibrium which is the expression of the immune system control on bacteria proliferation, but also of microorganism stimulations on immune cells.The sum of this process can promote both health and pathological situations such as obesity, inflammatory bowel diseases, cardiovascular problems and autoimmune conditions [133,134]   .Evidence on mice is demonstrating how commensal microbiota is necessary not only to develop functional but also licensed NK cells [135] .Findings have revealed that germ-free mice have a greater number of immature NK cells which are less active in response against viruses as NK cells of normal mice [135] are.Moreover, microbiota seems to have pivotal roles for both NK cells migration and education, producing complex signals thorough the cooperation of mucosal barrier cells which can induce tolerance or immune activation.It has been discovered that gut microbiota is able to down regulate the expression of NKG2D ligand on intestinal epithelial cells of mice [136] .When mice were treated with antibiotics, NKG2D ligand levels arise in the same way as in mice with celiac or colitis diseases [136] .Even though promising, the microbiome approach to understand NK cells biology in pathological conditions is only at its first steps and further studies are necessary to manipulate these lymphocytes for treatment purposes.
The idea to use NK cells to treat pathological conditions such as cancer has attracted researchers for years.However the new findings on NK cells biology seem to reduce the chances to manipulate these lymphocytes because, as described, NK cells activity results from the sum of activating and inhibiting signals and often it is difficult to determinate this threshold.Even if studies are focused principally on cancer treatment, recent promising approaches have arisen.International efforts are aimed at being able to expand endogenous NK cells in vivo using IL-12 and IL-15 [137] cytokines but are also they are focused on increasing the expression of NK cell activating ligands and death receptors on target cells [138] .Manipulation of chimeric NKG2D receptors on NK cell surface [139] seems to be effective against cancer cells and the transfer of purified donor NK cells have shown to generate long-term remission in patients with leukemia relapse [140,141] .Another strategy refers to enhance cell mediated ADCC (Figure 1) through the use of antibody combinations.Some examples are mogamulizumab, a monoclonal antibody which improve FcγRIIIa binding leading to increase ADCC NK cells activity [142] , but also the monoclonal antibody anti-CD137 potentiate rituximab ADCC mediated responses [143] .The block of the inhibitory receptor programmed cell death 1 (PD-1) in patients with multiple myelomas has been proved to increase NK cell cytotoxicity [144] .Moreover, growing studies show that the infusion of enriched NK cell populations, are resulting as safe and effective for treating cancer [140,145,146] .However, all these procedures are restrained by technical difficulties and costs which limit their use only in specialized centers.The detailed description of NK cell manipulation goes beyond the purpose of this review, but beside the methods cited, there are many newer laboratory approaches which have the purpose of taking advantage of the NK cells regulatory features to modulate immune responses paving the way for their use in vaccine, autoimmune and allergic diseases.

Conclusions and Future perspectives
As demonstrated above, NK cells play pivotal roles in immune response regulating other immune cells having diverse pathways.It has been described how these cells are involved in physiological maintenance of immune tolerance as well as in arising pathological autoimmune conditions.Disclosing NK cell interactions with adaptive immunity and microbiota is a fundamental process in developing a full comprehension of NK cells biology.Although evidence demonstrates NK cells activities have a great impact in both maintenance and rupture of immune tolerance, their precise role remains a dilemma.For these reasons, the actuation and production of consolidated immunotherapeutic approach to autoimmune diseases as well as cancer remains of difficult realization at the present time.Further investigations with a system biology approach should reveal and examine in depth NK cell relations in different autoimmune diseases considering not only the actions of human cells but also including the complexity of microbioa.Moreover, it is of fundamental importance to consider cell population interactions, as ILCs, and the signals produced in reaction to local stimuli focusing attention on local microenvironments.It is suggested that only with a multidisciplinary method it is possible to reveal the different NK cells actions improving our understanding on autoimmunity and self-tolerance preservation.

Figure 1 .
Figure 1.NK cells functions.NK cells functions are mediated by the constitution of immunological synapses.Every NK cell can interact with several cells at the same time and the consequent actions result from the signals which dictate the quality and intensity of NK cell response.A: Cytotoxicity activation.NK cells are able to lyse damaged cells such as virus infected or tumor cells.NK cell cytotoxicity activation can be managed in two different ways: 1-Lack of inhibitory signals.Injured cells can express activating ligands or can down regulate MHC-I ("missing-self theory") and other inactivating molecules as the result of pathological processes (virus or DNA damage).These surface modifications determine a lack or a diminished inhibitory signal which ultimately lead to activation of the NK cell cytotoxic activity which is characterized by the release of lytic granules towards the target.2-Antibody dependent cytotoxicity (ADCC).NK cells are able to lyse the target cells covered by antibodies through binding antibody Fc fragment thanks to the expression of FcγRIIIa.B: Cytotoxicity inhibition.NK cells are inhibited by different signals that derive from the engagement of inhibitory KIRs by HLA class I alleles and other molecules.If the stimulation of inhibitory receptors prevails, the NK cell activity is stopped.C: NK cell immunological interactions.NK cells can cross-talk with different cell types playing important roles in both innate and adaptive immunity.These lymphocytes can stimulate DCs maturation and activation but can also kill immature DCs influencing the presentation of antigens to T cells.Secreting cytokines, NK cell activate CD8 + T cells, increase antibodies production by B cells and induce proliferation of CD4 + T cells.Moreover NK can regulate macrophages enhancing or reducing their activities by secreting IL-10.(Mɸ: macrophages).

Figure 2 .
Figure 2. NK cells role in autoimmune diseases.NK cells seem to display a double role in autoimmune processes by different interactions with other immune cells.A: Protective role.NK cells could protect the organism from autoimmune reactions acting on different levels, in fact, they can control auto-antigens exposition clearing damaged cells such as viral infected cells, controlling thus some of the environmental factors.Furthermore, NK cells seem to be able to kill immature DCs and auto-antigens presenting DCs but also auto-reactive T cell clones.It has been demonstrated that NK cells can eliminate hyper-activated macrophages monitoring tissueinjuries and antigens presentation but can also enhance T regulators lymphocytes activities which down regulate auto-reactive T lymphocytes.By producing cytokines as IL-5, IL-10 or TGF-β, NK cells are able to directly modulate adaptive immune response reducing CD4 + T cell interaction with B cell which probably decreases the auto-antibodies production.B: Promoting role.The hypothesis of promoting role on autoimmune diseases demonstrates how NK cells may lose self-tolerance, inducing or participating in maintenance of autoimmunity.This process could be based on an altered expression of inhibitory KIRs which activate NK cell natural cytotoxic potential leading to direct tissue damage with a subsequent abnormal exposition to auto-antigens.Moreover, NK cells could be activated by healthy cells covered by auto-antibodies causing injuries and perpetrating autoimmunity.NK can also increase adaptive autoimmune process inducing DCs maturation in inflamed tissues, but also enhancing antibodies production, stimulating auto-reactive CD4 + T cells, promoting differentiation of naïve CD4 + T cells and increasing CD8 + T cells activation.NK cells are able to enhance macrophages activities such as antigen presenting cells role and cytokines.