In order to eliminate a pathogen, the immune system must recognize it and develop and adequate response to destroy it. The immune system has two types of mechanisms: innate and adaptive immunity. The innate immunity̕s strategy consists of recognizing a group of molecular patterns common to an entire family of pathogens.¹ Thus, all individuals within one species are born with innate ability to recognize and destroy numerous microorganisms immediately. Innate immunity is, therefore, able to fight infections from the beginning and during early stages with high efficiency (about 0-5 days). If these mechanisms fail to eliminate the infection, they at least at least they keep it under control until other mechanisms take place [1]. The first and most basic defense mechanism against infection are epithelia and mucous membranes, which are mechanical, chemical (defensins, lysozyme) and biological barriers against pathogens [2]. If microorganisms are able to cross these barriers and establish an infection, there are two pre-existing innate mechanisms that act immediately: humoral and cell - mediated immunity. Humoral immunity is mediated by macromolecules (as opposed to cell-mediated immunity) such as antibodies, complement proteins and certain antimicrobial peptides. Cell –mediated immuntity involves the activation of phagocytes, antigen-specific cytotoxic T-lymphocytes, and the release of various cytokines in response to an antigen [3]. All cells of the immune system need to be closely related to each other to develop a coordinated immune response that culminates in the elimination of the pathogen. Cells use two main ways to communicate: by direct contact with membrane molecules, and through cytokines synthesis. Many of the cytokines are produced in the first moment of cell activation, alerting cells with cytokine membrane receptors. Cytokines are molecules that often have only a local effect, taking part in antigen recognition and distance response, cytokines participate in regulation of the length and intensity of the innate and specific immune response, recruiting cells towards the conflict zone and inducing the generation of new cells from hematopoietic precursors [4]. Cytokines share functions with the so called host defense peptides (HDPs), proteins that act as effector molecules of innate immunity [5]. The cornea, as the outmost part of the eye, is constantly exposed to various agents potentially harmful to its surface and the internal structures of the eye, the reason why is endowed with various elements of innate and adaptive immunity, and is sheltered by anatomical structures, conjunctiva, and lacrimal system. The following will describe this in greater detail.

The tear film covers the ocular surface, and provides major refractive power of the visual system [32]. Other functions of the tear film include nutrition of the ocular surface, lubrication and a chemical barrier [33]. It forms a thin film layer of 8 μm thick; Although is typically said that is formed of three layers; the external or lipid layer, the central or aqueous layer and the inner or mucin layer, It is now recognized that the tear film is more a lipid boundary layer with aqueous phases incorporating differing concentrations of mucins throughout. Meibomian and Moll glands produce the lipid component [34], mainly wax esters, triglycerides, free fatty acids, as well as neutral diesters [35]. Lacrimal glands produce the aqueous component and goblet cells which are located in the conjunctiva, secrets the mucin and contains membrane associated glycoproteins [36]. Other components of the tear film are metabolites and electrolytes [37]. Interestingly, the proteins contained in the tear film take part in other processes, for instance, they work as antimicrobials, anti-inflammatories and also help in healing processes after trauma, as well as mechanical protection to the surface of the cornea [38]. Tears also transport carbon dioxide and oxygen, taking part the in ocular surface metabolism [39]. The meibomian glands are present in both the upper and lower lids in the tarsal plate and they are composed of many acini, formed by cells which secrete unique meibomian gland lipids, such as cholesterol, wax and cholesterol esters, which all the latter form part of the non-polar lipids, and account for 65% of the tear film lipids, as well as polar lipids (phospholipids and glycolipids) [40]. These substances are then excreted onto the ocular surface on the lid margin, where the mucocutaneous junction is located [41]. As mentioned, lacrimal glands are responsible of synthesizing the aqueous portion of the tear film. The main gland (orbital and palpebral portion) produces the reflex tear secretion onto the supero-lateral portion of the fornix, as well as accessory lacrimal glands produce the basal portion of the lacrimal film on the upper fornix [42]. The aqueous component accounts for 60% of the tear and it mainly contains water, electrolytes and proteins [43]. There have been identified dozens of different proteins contained in the tear, but the three main ones are: lysozyme, lactoferrin and tear-specific prealbumin (lipocalin) [44]. The lysozyme is an enzyme with antimicrobial function, as it destroys the glycosidic bonds between peptidoglycans in bacteria cell wall [45]. Lactoferrin protects the ocular surface from the effects of free radicals. The role of the lipocalins has not been clearly demonstrated, but they may have more than one function in the tear film; depurating the ocular surface from hazardous substances or aiding to spread the lipids on the tear film [46].

Mucins are hydrophilic glycoproteins which carbohydrate mass is of approximately 50-80%, Residues that are O-linked to serine and threonine, have in their protein backbone tandem amino acid repeats [47]. On the ocular surface, mucins can be associated with glycocalyx of conjunctival and corneal epithelium or with a mucous layer produced by the goblet cells [36]. The glycocalyx is a carbohydrate- based cover found on the surface of epithelia, to where mucous layers can get attached [48]. Mucins in the eye have multiple functions; by themselves, mucins lubricate the eye, indirectly they protect the epithelium from drying as they prevent tears from evaporating, they serve as another protective barrier against the hostile environment, they secure the aqueous layer of the tear film to the ocular surface, they protect from microbial invasion of the epithelium as they serve as a platform to hold other anti-microbial proteins to the eye [49,50]. Depending on the structure of their molecule, mucins can be of two different kinds; secreted or cell- surface associated mucins. Goblet cells are in charge of the synthesis of secreted mucins and of the properties of the mucous as well, while the membrane-spanning mucins have more to do with structural function [52]. Within the secreted mucins, two types can be identified, large gel-forming mucins and smaller nonpolymeric mucins. The main gel-forming mucin at the ocular surface is MUC5AC, which is localized in the goblet cells of the conjunctiva, whereas the nonpolymeric mucin MUC7 can be found in acinar cells of the lacrimal gland [51].

MUC1, MUC4 and MUC16 are examples of mucins expressed in the corneal and conjunctival epithelia. Other mucins such as MUC1, MUC2, MUC4, MUC5AC and MUC16, have been found in the tear film [47].

The term ocular surface is often employed in order to describe the assembly conformed by the cornea, the conjunctiva and the tear film which help maintaining visual acuity and also present interesting biochemical interactions that allow them to work as an immunological unit capable to respond to internal and external stimuli as well as modulating those responses in order to avoid exaggerated or chronic reactions [52]. Innate immunity provides a nonspecific surveillance system against corneal infection and is able to be the first form of protection, during the first minutes or hours, when the infection presents.

The ocular surface is formed by a variety of anatomical and functional components that include the bony orbit, eyelids, tears, corneal nerves, the corneal epithelium, leukocytes and cytokines [53,54]. The bony orbit and eyelids comprise the main anatomical structures that provide protection to the ocular surface, specially against traumatic events. Tears are secretions with the main function of lubricating and preventing the cornea from drying, flushing away foreign particles from the ocular surface, as well as distributing immunoglobulins (IgA and IgG) and antimicrobial proteins (lactoferrin, lysozyme, lipocalin and beta-lysin) to the ocular surface in order to prevent infections [55]. Cell-cell junctions of corneal and conjunctival epithelia complete the first line of anatomical barriers in the eye [56]. The corneal’ s innate immune system is comprised by many types of cells including epithelial cells, fibroblasts and Langerhan’s cells. Epithelial cells are in charge of the secretion of TNF-α, IL-1, IL-6 and IL-8.

Fibroblasts in the stroma may be in charge of the production of IL-1, IL-6, IL-8, TNF-α, and α-defensin as a way of after microbial infections; in addition, Langerhans cells in the cornea and conjunctiva are in charge of the adjustment of the B and T lymphocyte activity of the adaptive immunological response [53,57]. Toll- like receptors (TLR) are a group of fundamental molecules for the development of the innate immune response; so far 13 types of TLR have been recognized, TLRs 1 to 10 are present in humans, while TLRs 11 to 13 are unique to mice [58-60]. TLRs have extracellular and intracellular domains. The extracellular domain contains repeats that are rich in leucine (LRR: leucine-rich plates) while the intracellular domain is equivalent to the receptor of IL-1 (IL-1R: IL-1 receptor), and it may be named as the TIR: toll/il-1R domain [61].

Through the recognition of pathogen-associated molecular patterns (PAMPs), Toll like receptors are able to unleash immune responses. Structures that are preserved across the microorganisms of a given class and are vital for their survival, have become a great place of detection by the innate immune system since they enable a quick way to differentiate between non-self and self [60]. TLRs can be broadly divided by their location (intra or extracellular), or by the type of ligands they recognize (cell wall or genetic material) [54].

TLR2’s main function is to form heterodimers with TLR6 or TLR1 in order to recognize ligands from bacteria; TLR1/TLR2 heterodimers are able to recognize bacterial lipopeptides, including mycobacterial lipoprotein, meningococcal lipoproteins and triacylated lipoproteins [62-65]; TLR2/TLR6 heterodimer are able to recognize mycoplasma lipoproteins and peptidoglycan [66]; TLR2 can also act as an homodimer for the recognition of Gram-positive cell walls, lipotheicoic acid, mycobacterial lipoarabinomannan, zymosan and heat-shock protein [60,54,67,68]. TLR2 has also been suggested to be a Staphylococcus aureus innate receptor, functioning as a detector of Gram-positive bacterias in the cornea [64].

TLR4 is expressed in corneal and conjunctival cells, fibroblasts and dendritic cells; it is able to form complexes with CD14 and MD2 for the recognition of lipopolysaccaride (LPS) from Gram- negative bacteria cell walls [69]; its function requires several co- receptors, like MD2 and CD14 [65]. Unnecesary proinflammatory reactions due to the presence of commensal bacteria on the corneal surface are avoided by expressing minimal levels of TLR4 on the apical cells and the absence of MD2, one of the main co-receptors of TLR4 [66]. TLR5 is able to recognize flagellin, a major protein present in bacterial flagella [70]; it is a major sensor for the detection of bacteria that is Gram- negative and activates signalign pathways that liberate NF-kB and activate pro-inflammatory genes [71,72].

TLR5 are expressed on the surface of wing and basal corneal cells, different from superficial epithelial cells that do not express TLR5. Therefore, its activation only occurs when the barrier function of the cornea is compromised [70]. TLR3 is expressed by surface epithelial cells, where it acts as a receptor for viral double-strained RNA (dsRNA) [73], poly I:C and an analog of dsRNA [74]. Upon TLR3 stimulation NF-kB is activated, with the secretion of IL-6, IL-8, indicating that TLR3 is capable of unleashing an antiviral response.73 TLR7 and TLR8 have been implicated in the recognition of viral PAMPs, specifically single-stranded RNA (ssRNA), causing the virus infected macrophages to produce IFN-α and dendritic cells [54,75]. TLR9 is located in intracellular endosomal compartments, and specializes in the recognition of DNA with unmethylated CpG motifs from bacterial and viral genomes [76]. Activation of TLR9 induces a Th1 response with secretion of IL- 1277 IL-18 [54, 76, 77]. TLR activation leads to immune cell recruitment, cytokine release and the modulation of corneal healing. Activation of TLR pathways induces the specific expression of cytokines, and MHC molecules, helping an immune adaptive response to take place [78]. TLRs have several pathways: common adaptor molecule that is the primary-response protein 88 of myeloid differentiation (MyD88), TIR domain that comprises adaptor protein (this latter may also be known as TIRAP, MyD88-adaptor-like protein, or MAL), TIR-domain that has an adaptor that induces interferon β (also known as TIR-domain-containing- adaptor molecule 1, TICAM-1, or TRIF), and the molecule that is associated to the TRIF-related-adaptor (also known as TIR- domain-containing-adaptor molecule 2, TRAM or TICAM-2) [79].

The above have been described as the adaptor molecules, after this, the downstring signaling pathways are divided into MyD88-dependent and MyD88-non dependent. In order to produce signaling by TLR2 and TLR4, MyD88- dependent signaling is needed. This latter subsequently activates NF-kB [80,81]; For the induction of IFN-ß, TRIF works through the via of TLR3 and TLR4 MyD88-independent pathway [82].

The signaling pathway known as MyD88-independent/TRIF-dependent, is associated with activation by TRAM, and this takes places with the help of TLR4 that subsequently causes genes that are induced by IFN to express [83]. In order to have an adequate ligation of their PAMPs, receptors that are not related to TLR, for example MD2 and CD14 are used by TLRs. After the binding of this ligand, conformational adjustments take place with the purpose of enrolling downstream molecules for signaling.

The domain that is present in the C-terminus of MyD88, interacts with a TIR-domain and this causes the TLRs to recruit MyD88 which is the adapter molecule, and all of this as part of the MyD88- dependent pathway [84,85].

Through the death domain communication, the TLR complex receives through recruitment by MyD88, the IL-1R associated kinase (IRAK) and IRAK-4. Association of MyD88 to IRAK and IRAK, causes their phosphorylation and subsequent activation. Once IRAK is phosphorylated, it then separates from MyD88, and binds with the tumor necrosis factor receptor- associated factor 6 (TRAF6) [86]. Then, the TRAF6-IRAK complex binds to the TGF-β-activated protein kinase (TAK-1), which is associated with the TAK-1 binding proteins: TAB-1 and TAB-2. All these pathways trigger secondarily phosphorylation and activation of TAK1 [85].

As a result, the NF-kB-dependent transcription factor is activated after TAK1 once activated as well, phosphorylates the inhibitor of the NF-kB (IkB)- kinase (IKK) complex. This latter consists of IKKa, IKKb and NF-kB essential modulator (NEMO)/IKKg. TAK 1 can also causes the mitogen-activated protein and the stress associated protein to activate kinases (MAP and SAP respectively), for example: extracellular signal-related kinase (ERK), Jun N-terminal kinase (JNK) and p38. Pro inflammatory molecules such as IL-6, IL-8, IL-18, MIP-1 and TNF-α are produced secondarily after NF-kB is activated.

The transcription factor IFN regulatory factor 3 (IRF3) is phosphorylated due to activity of the MyD88-independent signaling. IFN-β type I is formed when phosphorylated IRF3 is translocated to the nucleus and activates its production. The production of IFN-β type I induces more production of IFN-α, IFN-β and the expression of IRF7 [85,87].

Despite its capability to produce and release proinflammatory cytokines, the ocular surface is also in constant contact with its comensal bacteria, mainly Staphilococcus epidermidis and Propionibacterium acnes, 88 which guides us to believe there must be negatively regulatory pathways for innate immunity, since a chronic inflammatory response in the cornea would lead to scarring and loss of visual acuity. Several molecules have been proposed to act as counter regulators, including MyD88 short, SIGIRR, Tollip, and ST2 [54]. These molecules confer the ocular surface unique characteristics that may contribute to the coexistence of comensal bacteria and TLRs [88]. Complete elimination of the offending microorganisms can only be achieved by the induction of adaptive immunity. TLRs initiate the inflammatory cascade by recruiting innate inflammatory cells to the sites of infection, like PMNs and NK lymphocytes; afterwards the arrival of monocytes and dendritic cells will initiate the adaptive immune response by capturing foreign antigens and presenting them to naive T lymphocytes by expressing MHC molecules [89, 90]. TLR agonists have proven to be effective for the treatment of some allergic, immunologic and infectious diseases in the ocular surface, TLRs are increasingly becoming therapeutic targets for the modulation of inflammation in the ocular surface, avoiding or becoming alternatives to broadly employed anti- inflammatories like corticosteroids [57]. The cornea can be considered an immune privileged tissue, a term developed in order to explain the high success rate of corneal transplantation [91]. This situation was at first explained by the lack of vasculature and lymphatic drainage in the cornea, resulting in a delay in the traffic of antigen presenting cells (APC) to lymphatic nodes, and the barrier the lack of vasculature represents for effector cells. However, recent research has revealed the process to be more complex and active, rather than passive; diverse endocrine, neural and immunological factors are essential for the preservation of the ocular surface and normal visual function [52]. Local and systemical modulation is necessary to maintain immunological homeostasis. Intraocular regulation is achieved in part due to the pigmented epithelial cells of the iris by producing immunosuppresive molecules like CD86, PD-L1 and CTLA-2α, that are capable of inducing Tregs. Pigmented cells also release TGFß, an immunoregulatory cytokine, antigen presenting cells in the ocular tissue are exposed to it and generate antigen-specific Tregs that migrate to the spleen and establish immune tolerance to those antigens, this process is known as anterior chamber immune deviation (ACAID) [92]. Neural pathways also have an important paper in inducing inflammatory response. Presence of neuropeptides inside the aqueous humor possesses immunoregulatory characteristics, while the presence of sensory and sympathetic nerve fibers in the cornea allows the maintenance of ocular immune privilege by increasing the le vels of TGFß [93]. Some authors have proposed that parasympathetic nerve fibers could also have an important paper on the ocular surface immunoregulation by increasing the levels of VIP and somatostatin [94].

There are many studies indicating that a myriad of molecules such as neuropeptides, mucins, pattern recognition receptors, among others are involved in the very complex immune system of the ocular surface just described. Taken together these results, reinforce the idea that the ocular surface is a versatile epithelial barrier.