Introduction
Dry eye disease (DED) is a multifactorial condition of the ocular surface, characterized by chronic inflammatory damages of the cornea and conjunctiva, along with the involvement of Meibomian and lacrimal glands, and neurosensory system innervating the ocular surface. The key underlying pathophysiological process driving the disease is due to the loss of immune homeostasis at the ocular surface, with subsequent disruptions of the tear film stability, corneal epithelial barrier, and corneal sensation. DED can be broadly classified into two categories based on the lack of aqueous tears or not—aqueous-deficient form (with reduced tear quantity) and evaporative form (with reduced tear quality), , and accordingly various animal models have been created to mimic DED through decreasing the tear production or increasing the tear evaporation. It is worth to be noted that the two types of DED are not mutually exclusive, and one model that primarily falls into one category may still possess some features of the other. In fact, coexistence of deficiencies of tear quality and quantity is not uncommon in human patients with DED, and recent report from the Dry Eye Work Shop (DEWS II) has advocated a continuum classification scheme to highlight such overlap between the two primary types of DED. Therefore, it is suggested that no matter how the disease is initially induced, characteristic features of both types of DED may occur as the disease progresses, and common pathophysiological processes are shared among various etiologies of DED in both human and animal models.
It is well known that continued inflammation plays a key role in DED, and significant advancement has been achieved in the past 2 decades in our understanding of the precise pathogenesis, particularly the immune mechanisms underlying the disease, primarily through the valuable animal models including the transgenic mouse models of Sjögren’s syndrome and the popular desiccation stress-induced non-Sjögren’s DED mouse models that were established independently by both Pflugfelder (Baylor College of Medicine) and Dana (Harvard Medical School) groups in early 2000s. Mouse models are the most popular in corneal research, including DED, although rat, rabbit, and dog DED models have also been reported. Mice are particularly useful to study the disease pathogenesis given their similar ocular surface structures with humans, well-defined immunogenetics in inbred strains, abundant availability of reagents and various transgenic and knockout strains, as well as small size and ease of husbandry. Anatomically, mouse cornea has similar layers to human cornea, including epithelium, stroma, posterior limiting lamina (Descemet’s membrane), and endothelium, despite the presence of anterior limiting lamina (Bowman’s membrane) in mice is questionable ( Fig. 4.1 ). However, major discrepancies also exist—the diameter and thickness of mouse cornea are much smaller than human cornea. Unlike the oval shape in human, mouse cornea is round in shape and its diameter measured from limbus to limbus is about 2.6 mm in adult (6–8 weeks) C57BL/6 strain, the most commonly used strain for murine DED model. The central corneal full thickness in adult C57BL/6 is about 140 μm, higher than peripheral thickness of about 90 μm; the individual epithelium and stroma layer shows similarly thicker center than periphery. In contrast, human cornea is thinner in the center (535 ± 20 μm) than periphery (657 ± 71 μm). Clinically, corneal epitheliopathy is the most evaluated sign of DED, and is examined by corneal fluorescein staining (CFS), which stains areas of epithelial defect or where the epithelial barrier is lacking. , , Corneal epithelial barrier in both mouse and human is similarly formed by desmosomal junctions, hemidesmosomes, and basement membrane, except there are more layers of epithelial cells in mouse than in human, , and thus CFS is similarly used for evaluating disease severity in mouse DED models. In addition to epithelial layer, corneal stroma is also critically involved in the pathogenesis of DED, as it is the principal site where bone marrow–derived immune cells reside in healthy cornea (with a few residing in epithelial layer) which are in resting status in normal but become activated in DED. , The mouse stroma is similarly arranged into lamellae with scattered keratocytes as observed in human, with some different fiber organization patterns in the anterior part of the stroma. The corneal resident immune cells are characterized as primarily CD11b + CD3 − CD19 − myeloid but not lymphoid lineage cells, including dendritic cells (Langerhans cells within epithelium), macrophages, and monocytes. , Furthermore, the distribution pattern and functional status of corneal immune cells are consistent between human and mouse: significantly more myeloid cells are localized in the corneal periphery (throughout the entire stroma) than in the center (primarily in anterior stroma), and these cells represent phenotypically “immature” status that contributes to the immune homeostasis in normal cornea. , , , Lastly, corneal nerve is substantially involved in DED pathogenesis, due to its critical roles in corneal pain sensation, epithelial integrity, and release of neuroinflammatory substances. Nerves enter the corneal stroma at the limbal circumference from the ophthalmic branch of the trigeminal ganglion, and extends anteriorly toward corneal epithelium, forming the subbasal nerve plexus that supplies the overlying corneal epithelium. , A major difference in corneal nerves between mouse and human is that the nerve fibers distribute deeper in mouse (closer to endothelium) than in human. In summary, it is important to be aware of both similarities and differences in histological structures and cellular components between mouse and human cornea for us to correctly extend research findings from mouse models of DED to human patients.
Clinical Findings in Animal Models of Dry Eye Disease
The common ocular symptoms in DED patients include ocular discomfort, visual disturbance, burning sensation, or pain. , However, subjective symptoms are hardly assessed in animal models due to the issue of feasibility. On the other hand, the recommended diagnostic tests in patients by the most recent DEWS II reports include tear film break-up time, tear osmolarity, tear volume, and ocular surface fluorescein staining. All of these objective criteria, through various combinations, have been used in evaluating DED-related signs in animal models.
Desiccating Stress-Induced Dry Eye Disease
Desiccating stress, including environmental and pharmacologic ways, has been a popular method to induce DED in animals since it was initially validated in mice 2 decades ago. , The advantages of this model over others include relatively localized eye disease with minimally systemic effects, use of wild-type animals without genetic modifications, reliable and reversible (i.e., treatable) disease induction. Initial efforts to test the effect of desiccating stress on cornea were made by preventing eye blinking through placement of lid specula in rabbits for 2 h, which led to methylene blue staining on cornea, a sign of corneal epithelial defect, and corneal epithelial thinning demonstrated by scanning electron microscopy. However, this model presents a very acute process lasting only hours, and animals have to receive anesthetics which themselves can affect tear secretion. Other researchers used a pharmacologic way by topical application of 1% atropine sulfate, an anticholinergic agent which inhibits aqueous tear secretion by lacrimal glands, in rabbits three times daily for 5 days, and they observed significantly decreased tear production and increased CFS as early as day 2. Pflugfelder group (Baylor College of Medicine) later combined continuous airflow with transdermal scopolamine patch, an inhibitor of muscarinic acetylcholine receptors, to induce DED in female CBA mice. After 4-day’s stress, mice showed significant reduction of tear secretion using cotton thread test (CTT) and increased punctate CFS; however, environmental desiccating stress alone (continuous airflow alone without scopolamine) generated in this model through an air fan placed at 6 inches in front of the mice’s cage did not effectively induce significant ocular surface changes, suggesting that the environmental desiccating stress created in this model may not be adequate. Subsequently, Dana group (Harvard Medical School) optimized several key environmental factors by creating a more stable and efficient Controlled-Environment Chamber (CEC) which provided continuous dry airflow with low humidity (via filtering through desiccant columns) directly into the chamber where mice were housed (via airlines connected to an air pump outside the chamber and regulated by a flow meter) as well as a constant temperature of 21°C to 23°C inside the chamber. All these environmental parameters were monitored by a probe. Combined with subcutaneous scopolamine injection, the CEC led to significantly decreased tear secretion and increased CFS in female BALB/c mice as early as day 3 post desiccating stress and throughout to the end of observation at day 28. Further studies from Dana group demonstrated that C57BL/6 mouse strain showed more severe tear reduction than BALB/c strain under the stress, while the CFS severity remained comparable between the two strains ; this is probably related to the differential biased immune response in C57BL/6 (prone to Th1 response) and BALB/c (prone to Th2 response) that are relevant to the pathogenesis of DED (to be discussed later in the following sections). This study has made C57BL/6 the most frequently used mouse strain in basic and translational DED research. , Since the beginning of the development of DED models, female animals were exclusively selected given the fact that the significant majority of human patients suffering from DED are women, and a recent study using the combined environmental and pharmacologic desiccating stress model confirmed that female C57BL/6 mice presented with significantly higher CFS scores and lower tear production than male counterparts. Since the creation of CEC, continuous efforts have been made to improve the stability of the low humanity inside the chamber including the use of an intelligent dehumidifying device. , By replacing the air-drying desiccant columns with a digitally controlled dry cabinet inside which the circulating air was supplied from atmosphere air that was first dried via membrane dryers before entering the chamber ( Fig. 4.2 ), the daily variation of the humidity observed in the original CEC system was significantly reduced, and thus a constant low humidity below 15% was achieved throughout different seasons in a year. Additionally, the dry air is continuously pumped into the mouse cage through four air lines connected to two opposite walls of each cage. With such improvement, mice developed a severe corneal epitheliopathy evidenced by all quadrants thick punctate CFS with 14-day’s desiccating stress; and importantly, after mice were transferred to nondesiccated vivarium without any further pharmacologic desiccating stress, the epitheliopathy gradually regressed to lower levels, but never normalized, for more than 4 months observation period, despite the resolution of aqueous tear deficiency. Thus, for the first time, a chronic DED model was established, which is critical because DED is generally encountered as a chronic disorder (months to years) in the clinical setting, and experimental data derived from a chronic model are more related to humans than that from an acute model.
Scopolamine or other anticholinergics are able to effectively suppress tear production by lacrimal glands via blocking acetylcholine receptors and thus induce DED without concomitant environmental desiccating stress. , However, there have been concerns regarding the use of the anticholinergics due to the existence of an independent, nonneuronal cholinergic system in lymphocytes. Animal study has shown that in vivo treatment with systemic scopolamine in C57BL/6 mice leads to general enhancement of Th2 (type 2 T helper cell) and Treg (regulator T cell) responses but inhibition of Th17 (type 17 T helper cell) response, which are consequences of direct pharmacologic effects of scopolamine on T cells but not DED pathogenesis relevant. Therefore, pharmacologic desiccating stress may not be a suitable model for DED pathophysiology investigations, and care should be taken in interpreting research findings in those models created through systemic acetylcholine receptor blockade. In fact, the improved, digitally controlled CEC system has been shown to effectively induce both acute , , and chronic DED in mice without the use of scopolamine. These DED mice exhibit consistently high CFS scores and ocular pain, a common symptom seen in human patients, as demonstrated by increased eye wipe response to topical hypertonic saline stimulation (R. Dana, unpublished data). Ocular surface sensory nerves express polymodal nociceptors, mechanonociceptors, and cold thermoreceptors, and all of them contribute to pain sensation with the cold thermoreceptors also functioning to regulate basal tear production and blinking. The hyperosmolarity formed by hypertonic saline at the mouse ocular surface activates both transient receptor potential cation channel subfamily V member 1 (TRPV1) channel (expressed by substantial proportions of corneal nerve endings and mediating polymodal nociceptor sensory transduction) and transient receptor potential cation channel subfamily M member 8 (TRPM8) channel (expressed by relatively smaller proportions of corneal nerve endings and mediating cold thermoreceptor sensory transduction).
Surgical or Medical Destruction of Lacrimal Gland
The lacrimal gland is the main contributor to the aqueous layer of the tear film by secreting water, electrolytes, and proteins. Mechanically obstructing or removing the lacrimal glands has thus been used to induce aqueous-deficient DED. After lacrimal gland duct was closed via cauterization in rabbits, tear film osmolarity was progressively increased, but there was no changes in Schirmer’s test probably due to the compensatory secretion from the accessory lacrimal glands. , Similarly, gland obstruction achieved by excretory duct ligation in mice led to increased CFS. Surgical removal of bilateral extraorbital lacrimal glands is a more aggressive way to induce severe DED. Both rats , and mice were used to establish this type of model, and animals showed decreased aqueous tear production and increased CFS scores, more severe than that observed in desiccating stress-induced DED. Consistent with the sexual predisposition in desiccating stress-induced models, extraorbital lacrimal gland excision led to more severe corneal epitheliopathy and ocular pain (measured by eye wipe response to capsaicin, an activator of TRPV1 channel) in females than in males. , , Cold allodynia demonstrated by eye closing response to innocuous cold air flow was also reported in the mouse model of extraorbital lacrimal gland excision. In addition, combined extraorbital and intraorbital lacrimal glands excision resulted in a comparable or more severe clinical DED. ,
Systemic delivery of scopolamine or topical application of atropine eye drops have been described above as pharmacologic desiccating stress via inhibiting lacrimal gland function. Additional methods of medical inhibition of lacrimal glands have also been reported to induce DED, including direct intralacrimal gland injection of toxins or cytokines. Female mice receiving one single intralacrimal gland injection of botulinum toxin-B, a well-known antiacetylcholine toxin, presented with significantly decreased tear production as early as day 3 postinjection and lasting for 4 weeks; after 4 weeks, despite normalized tear production, the animals showed persisting, significantly increased CFS, suggesting that this model represents another form of chronic DED with similar clinical findings from CEC-induced chronic DED. On the other hand, injection of a neurotrophin receptor antibody-conjugated saporin toxin into lacrimal glands of rats led to the damage of nerves within the glands and decreased eye wipe response to corneal menthol stimulus (assessment of polymodal nociceptor-mediated pain); however, basal tear production and eye wipe response to corneal application of capsaicin (assessment of cold nociceptor-mediated pain) remained normal in animals. Such corneal hypoalgesia observed in this saporin toxin model was also a commonly seen symptom in DED patients. , In addition to toxins, injection of proinflammatory cytokine IL-1α into lacrimal glands was used to induce DED. A single injection led to rapid reduction of basal tear secretion in both C57BL/6 and BALB/c strains; however, this effect disappeared spontaneously by day 13 in C57BL/6 and by day 7 in BALB/c, respectively. Lastly, an autoimmune rabbit model was reported by intralacrimal injection of in vitro–stimulated autologous peripheral blood lymphocytes with lacrimal epithelial cells. Animal developed decreased tear production (by Schirmer’s test) and tear stability (by tear break-up time) and increased corneal rose Bengal staining. ,
Genetic Manipulation–Induced Dry Eye Disease
Various types of selectively inbred or genetically modified mice demonstrate DED phenotypes in addition to other co-existing systemic immunoinflammatory disorders, and most of them are generated on the C57BL/6 background.
Mouse models of Sjögren’s syndrome
Mouse models of Sjögren’s syndrome are among the earliest animal models established for DED study, and these models show common lymphatic infiltration in both lacrimal and salivary (submandibular) glands, termed dacryoadenitis and sialadenitis, respectively. Nonobese diabetic (NOD) mouse, the most commonly used Sjögren’s syndrome model, was developed through a highly selective inbred process. They develop spontaneous insulitis leading to type 1 diabetes, which occurs before the development of exocrinopathy, and thus the Sjögren’s syndrome in this model is considered to be secondary. , In contrast to human patients and wild-type animal DED models, autoimmune dacryoadenitis in NOD mice is more prone to develop in male than female, evidenced by much earlier onset and higher incidence of dacryoadenitis in male animals. Clinical DED develops subsequent to the lymphatic infiltration of lacrimal glands, and the relevant findings in NOD male mice include decreased tear production and tear film break-up time, as well as increased CFS. With aging, NOD mice showed not only increased corneal permeability measured with the fluorescent Oregon green dextran dye but also increased tear volume measured with cotton thread technique. Further studies have developed new strains of NOD mice that develop glandular infiltrates but not overt diabetes, including C57BL/6.NOD-Aec1Aec2 and NOD. H2 b ; however, no significant change in corneal permeability was detected in either strain. ,
MRL/lpr mouse is another important model of Sjögren’s syndrome, which carries the lpr (lymphoproliferation) mutation leading to defective expression of Fas, a critical molecule mediating cell apoptosis. Distinct to NOD mouse, MRL/lpr mouse has a female predisposition to develop dacryoadenitis, accompanied with systemic lupus erythematosus and arthritis. Despite similar focal lymphocytic infiltrates in lacrimal and salivary glands as that in human Sjögren’s syndrome, the clinical tear production as measured by Schirmer’s test in MRL/lpr mice was nearly normal. However, CTT, a more accurate and sensitive measurement of tears in small animals like mice, showed reduced aqueous tear production in MRL/lpr mice.
CD25 is the cytokine IL-2 binding receptor alpha chain and serves as the limiting factor of IL-2 signaling, which is critical for the survival and function of immunoregulatory T cells that essentially suppress abnormal immune activation. It is thus expected that absence of CD25 signaling will lead to inappropriate immune response, including autoimmunity to self-tissue. In fact, CD25 knockout (KO) mice spontaneously develop Sjögren’s syndrome-like lymphocytic infiltration in exocrine glands, including the salivary and lacrimal glands ; in addition, they develop autoimmune inflammatory bowel disease and hemolytic anemia that leads to early mortality. The development of dacryoadenitis in CD25 KO mice was observed as early as 8 week of age without sex predilection, and it got worse when the mice became aged. The ocular surface findings in CD25 KO mice include increased corneal staining with Oregon green dextran dye and decreased corneal surface smoothness. ,
Thrombospondin-1 (TSP-1) is a matricellular protein that activates latent TGF-β1, an important antiinflammatory cytokine. Thus, mice deficient in TSP-1 develop inflammatory infiltrates in multiple organs, including in lacrimal glands that leads to a Sjögren’s syndrome-like pathology, but in a milder degree than TGF-β1 KO mouse. In addition, unlike TGF-β1 KO mouse which has a shortened life span, TSP-1 KO mouse exhibits a normal life span, making them more suitable for scientific study. TSP-1 KO mice of both sexes develop progressively increased CFS and even dry crusty eyes in severe cases. There is no significant changes in tear production.
Autoimmune regulator (Aire) -deficient mice develop spontaneous multiorgan autoimmune disorders, involving retina (uveitis), the salivary and lacrimal glands (Sjögren’s syndrome), and others. Accompanied by inflammatory destruction of lacrimal glands in Aire -deficient mice, stimulated tear production was significantly reduced and corneal lissamine green staining was profound. , Other Sjögren’s syndrome–related models associated with lymphocytic infiltration in lacrimal glands have been described such as NZB/W F1 mouse, TGF-β1 knockout mouse, and aly/aly mouse , ; however, impact on ocular surface tissues in these mouse strains is unclear due to the lack of relevant reports.
SPDEF knockout and mucin knockout mice
Conjunctival goblet cells are the major source of ocular surface mucins which are critical to maintain the homeostasis of the tear film, such as MUC5AC the most abundant mucin in the tear film. Decreased number of conjunctival goblet cells has been identified in multiple animal models of DED and is used as one of the histological features for DED. , SAM-pointed domain epithelial-specific transcription factor (SPDEF) is the transcription factor essential for goblet cell differentiation in mucosal epithelium, and Spdef KO mice which lack conjunctival goblet cells show significantly increased CFS and tear volume (measured by CTT) that progress with aging, suggesting an evaporative type of DED. Relatedly, Muc5ac KO mice showed decreased tear break-up time and increased CFS without aqueous tear production change, although another study did not find increased CFS in either Muc5ac KO or Muc5b KO mice.
Neurokinin-1 receptor (NK1R) knockout mouse
NK1R is the preferred receptor for substance P (SP), a neuropeptide mediating corneal pain and promoting corneal inflammation when upregulated in diseased status. , , However, basal levels of SP in tears may play a critical physiological role in maintaining ocular surface homeostasis, and mice genetically deficient for functional NK1R showed reduced level of aqueous tear secretion, without significant increase of CFS.
Animal Models of Meibomian Gland Dysfunction
The Meibomian gland is a specialized sebaceous gland located in the eyelids. The gland produces a lipid secretion called meibum that constitutes the lipid layer of the tear film and prevents the underlying aqueous layer from evaporation, thus maintaining the homeostasis of the tear film. Meibomian gland dysfunction (MGD) is one of the major causes of DED, and various animal models of MGD have been developed, including rabbit models and a variety of transgenic mouse strains.
Obstruction of Meibomian gland
Mechanical obstruction of Meibomian gland duct resulting from epithelial hyperkeratinization and/or increased viscosity of the secretion (meibum) is considered the most common cause leading to MGD. Using larger animals such as rabbit, electrocauterization was used to cause mechanical damage to Meibomian gland orifices and subsequent keratinization and fibrosis of orifices, leading to the blockade of the gland. Animals showed significantly increased tear osmolality without significant changes of CFS or aqueous tear production except a short temporary reduction by CTT at day 1 postcautery. , In a similar rat model of Meibomian gland orifice obstruction, animals showed significantly reduced tear break-up time, increased CFS, but stable tear production by Schirmer’s test. In addition, earlier studies using chronic topical application of 2% epinephrine over a period of months to 1 year led to orifice plugging in rabbits, but there is no tear film or CFS data available. ,
Mutant and transgenic mouse models
HR-1 hairless mice were used to develop MGD model by feeding them with HR-AD diet (a special diet with limited lipid content). In addition to atopic dermatitis-like symptoms, these mice presented markedly plugged orifices and a toothpaste-like meibum. Apolipoprotein E knockout ( ApoE −/− ) mice are characterized by increased blood cholesterol levels, and they were found to present with the typical clinical MGD manifestations of Meibomian gland dropout (progressive loss) and increased CFS. X-linked anhidrotic-hypohidrotic ectodermal dysplasia (EDA) mice resulting from Eda gene mutation completely lacks Meibomian gland, and they develop corneal neovascularization, keratitis, ulceration, and keratinization, as well as blepharitis and conjunctivitis. Relevant DED signs in this extreme MGD model include increased CFS and decreased tear break-up time, while aqueous tear secretion is normal. Other transgenic mouse models leading to defective Meibomian gland development ranging from gland absence to atrophy or dystrophy include ectoderm-targeted overexpression of the glucocorticoid receptor (GR), epithelial cell–conditional fibroblast growth factor receptor 2 (FGFR2) knockout, acyl-CoA:cholesterol acyltransferase-1 (ACAT-1) knockout, conditional deletion of Krüppel-like factor (KLF), homeobox transcription factor Barx2 knockout, and among others, and the DED-relevant ocular surface signs have not been evaluated in any of them.
Age-related Meibomian gland dysfunction
Studies of human and mouse Meibomian glands have shown decreased acinar cell proliferation and increased glandular atrophy during aging, which leads to Meibomian gland dropout and abnormal lipid excretion without epithelial hyperkeratinization, referred to as age-related Meibomian gland dysfunction (ARMGD). Meibomian gland dropout is seen in wild-type C57BL/6 mice over 1 year of age, and ARMGD has been documented in 2 years old C57BL/6 mice showing increased Meibomian gland dropout and CFS, and decreased tear secretion. , However, the clinical DED signs in aged mice are not entirely due to the changes of Meibomian glands, other changes in lacrimal glands, immune homeostasis, and hormone levels also critically contribute to the DED phenotypes in the aged. ,
Other Models
Spontaneous canine DED
Spontaneous DED is commonly seen in dogs, and the incidence is estimated about 35% with more prevalent in the aged and equally involving male and female. The disease is considered mainly autoimmune-driven and tends to be more severe than that observed in humans. It is diagnosed with aqueous tear deficiency by Schirmer’s test. This model has been used to test the therapeutic effect of topical cyclosporin A eye drops, the first and one of only two US FDA-approved drugs currently for treating DED patients.
Benzalkonium chloride–induced DED
Benzalkonium chloride (BAC), in its low concentration (<0.02%), is a commonly used preservative in ophthalmic medications. In patients using BAC-preserved eye drops, DED-associated symptoms and signs are more prevalent. Rabbits treated with topical 0.1% BAC twice daily for 14 days exhibited significant reduction of tear production (Schirmer’s test) and increase of CFS and rose Bengal staining. , Female C57BL/6 mice treated with topical 0.075% BAC twice daily for 7 days showed significantly increased corneal staining with Oregon green dextran dye and decreased tear production measured by CTT.
Androgen deficiency–related DED models
As seen in most autoimmune disorders, DED predominantly affects women than men, and the female sex is a significant risk factor for the development of DED with sex hormones believed to be critically involved. Androgen deficiency in castrated rabbits was associated with lipid content alterations in the Meibomian gland. Male rats and rabbits with bilateral orchiectomy showed significantly reduced tear production measured by Schirmer’s test and shortened tear break-up time. , In addition, ovariectomy in female mice led to decreased estradiol and testosterone levels, as well as DED signs of reduced tear production and increased CFS.
High fat diet–induced DED
A recent study reported that C57BL/6 mice subjected to high fat diet for up to 4 months developed overweight, hyperlipidemia, and DED-like clinical changes, including deceased tear production and increased Oregon green dextran staining, which were correlated with oxidative stress at both ocular surface and lacrimal glands. ,
Ocular surface hyperosmolarity–induced DED
Tear hyperosmolarity (≥308 mOsm/L) is frequently detected and used as one of the diagnostic tests in DED patients. Tear hyperosmolarity can result from decreased aqueous tear production or increased tear evaporation. Topical application of hypertonic saline (900 or 3000 mOsm/L, four times daily for 5 days) to the ocular surface of BALB/c and C57BL/6 mice did not lead to significant changes in CFS or tear production, but caused decrease in corneal nerve density and led to immune cell activation. , Another report using much longer stressing time (500 mOsm/L, six times daily for 28 days) found that mice developed significantly increased CFS.
Pathophysiology of Dry Eye Disease Learned from Animal Models
Characteristic pathologies in ocular surface of DED animals include (i) changes in corneal epithelial thickness/cell shape, (ii) decreased numbers and atrophy of conjunctival goblet cells, , , (iii) apoptosis of corneal and conjunctival epithelia, and (iv) basal acinar cell proliferation and altered lipid production in Meibomian gland. The central mechanisms leading to these pathological consequences are believed to be driven by a vicious cycle of ocular surface inflammation that is initiated by various intrinsic or extrinsic triggers, such as desiccating stress, and thus the underlying immunopathogenesis is discussed in this section.
Immune Homeostasis of Normal Ocular Surface
The ocular surface comprises of a continuous mucosal lining of cornea and conjunctiva, extending to the mucocutaneous junctions of the lid margins. Despite being constantly exposed to outside environment, the ocular surface remains integrated and uninflamed while keeping its visual function. This ability to maintain immune homeostasis is mediated through both physical barrier function and active mechanisms, encompassing epithelium, stroma, nerve, and resident immune cells. The avascular nature of normal cornea is essential for its transparency and the “immune privileged” status by creating an access barrier for circulating immune cells to enter the site. To maintain the avascularity, corneal epithelium constitutively expresses soluble vascular endothelial growth factor receptor-1 (sVEGFR-1) to inhibit VEGF-A-mediated new blood vessel formation (hemangiogenesis), and VEGFR-3, serving as a trap, to prevent VEGF-C- and VEGF-D-mediated new blood and lymphatic vessels formation (lymphangiogenesis).
Normal cornea has no T or B lymphocytes, but is endowed with a significant population of CD11b + CD11c − macrophages/monocytes in the deep stroma, along with some CD11b + CD11c + dendritic cells in the anterior stroma and CD11b lo/− CD11c + Langerhans cells in the epithelium. , , These cells are collectively believed to be capable of functioning as antigen-presenting cells (APCs) in immune response, and they are phenotypically “immature” characterized as low expression levels of MHC class II (MHC-II) along with absence of costimulatory molecules B7 (CD80 and CD86) and CD40. These immature APC predominantly reside at corneal periphery and decrease in number gradually toward the corneal center. The immature status of corneal-resident APC along with absence of lymphocytes in normal cornea contributes to the immune quiescence by not only avoiding effector cell activation but inducing immune tolerance. ,
Corneal epithelium also constitutively expresses a variety of critical immunoregulatory factors, including programmed death-ligand 1 (PD-L1), Fas ligand (FasL), pigment epithelial-derived factor (PEDF), and thrombospondin-1 (TSP-1). , PD-L1 is a newer member of B7 family, and its ligation with the receptor programmed death (PD)-1 on activated T cells leads to suppression of T cells. PD-L1 KO mice show spontaneously significant T-cell infiltration in cornea. In addition, PD-L1 actively inhibits corneal hemangiogenesis and thus contributes to corneal avascularity. Similarly, FasL, a member of tumor necrosis factor (TNF) family, can lead to apoptotic cell death by binding to its receptor Fas that is expressed by a variety of cells and tissues, including T cells, and prevent neovascularization. , Two forms of FasL have been identified including the antiinflammatory, soluble form (sFasL) that can antagonize the function of the other proinflammatory, membrane-bound form (mFasL). Although sFasL is the predominant form of FasL expressed in the retina of mice, it remains unclear what form(s) of FasL that is constitutively expressed in the cornea. Both PD-L1 and FasL constitutively expressed in corneal epithelium may trigger apoptosis of invading effector T cells that infiltrate in the cornea in response to inflammation. PEDF is a ubiquitously expressed glycoprotein belonging to the serine protease inhibitor family. TSP-1 is a major activator of latent TGF-β, and only activated TGF-β can serve as an antiinflammatory cytokine. , Normal corneal epithelium–derived PEDF and TSP-1 have both been shown to potently suppress APC activation. , Both factors exert antiangiogenic function , with PEDF showing additional neurotropic roles.
The cornea is among the most densely innervated tissues, and corneal nerves play a crucial role for ocular surface homeostasis by protecting the cornea from irritants (by regulating tear secretion and the blink reflex) and by secreting a variety of neuropeptides that are essential for epithelial and stromal cells. Healthy innervation in cornea has been shown to regulate corneal epithelial cell and stem cell survival, , and suppress corneal neovascularization. Corneal neuropathy has been observed in DED patients and animal models. , , , Specifically, among various nerve-derived factors, neuropeptides SP and vasoactive intestinal peptide (VIP) are constitutively secreted by nerve endings in normal cornea, with SP contributing to corneal epithelial homeostasis and VIP playing antiinflammatory functions. ,
Innate Immunity Activation
The immune homeostasis of ocular surface is disrupted in DED, mediated by both innate and adaptive immunity. Innate response serves as the immediate, nonspecific reaction to various insults, and the involving components include mucosal barriers, cytokines, chemokines, and innate immune cells.
Innate inflammatory cytokines release by ocular surface tissues
Desiccating or hyperosmolar stress on ocular surface, either direct or consequent to lacrimal or Meibomian glands damage that results from extrinsic insults or intrinsic changes, can be the initiating factors driving the development of DED by triggering immunoinflammatory cascades at the ocular surface. Stressed ocular surface epithelial cells quickly upregulate the levels of activated mitogen-activated protein kinases (MAPK), including c-jun N-terminal kinases (JNK)-1/2, extracellular-regulated kinases (ERK)-1/2, and p38 in hours, , , which may subsequently activate downstream kinases and transcription factors such as NFκB, leading to the early expression of proinflammatory cytokines TNF-α, IL-1β, and IL-6 at the ocular surface. , , , In addition, desiccating stress can elicit the cellular signal of damage-associated molecular patterns, which activate toll-like receptor 4 (TLR4)—a critical innate immune mechanism—in cornea, demonstrated by translocation of TLR4 in epithelial cells (from cell inside to cell surface) and upregulation in stroma cells. TLR4 activation further leads to downstream caspase-8 activation facilitating the assembly and activation of inflammasomes NLRP12 and NLRC4, thereby promoting the activation and secretion of proinflammatory cytokines such as IL-1β. In addition to TLR4-mediated pathway, increased reactive oxygen species upon desiccating stress exposure induced activation of caspase-8 and NLRP3 inflammasome, further promoting the production of bioactive IL-1β. Both IL-1β and TNF-α have been shown to promote corneal expression of matrix metalloproteinase-9 (MMP-9), , a critical proteolytic enzyme cleaving epithelial basement membrane components and tight junction proteins (such as ZO-1 and occludin) and thus leading to corneal barrier disruption. ,
Innate immune cell infiltration and activation at the ocular surface
Early infiltration of CD11b + monocytes and macrophages in DED corneas is a hallmark consistently found in various animal models. , , Influx of these innate inflammatory cells relies on the chemotactic gradient between the periphery and corneal tissues created by higher expression of various chemokines on the ocular surface in DED, including macrophage inflammatory protein 1α (MIP-1α, or CCL3) and MIP-1β (CCL4). Chemokines are small molecular weight cytokines with chemoattractant properties that serve an essential function in immunity by coordinating the trafficking of immune cells between different anatomic sites. In some cases, multiple chemokines may interact with the same chemokine receptor. Increased levels of these chemokines at ocular surface in DED can lead to tissue-specific recruitment of peripheral circulating monocytes that express corresponding specific receptors, including those expressing CCR1 for chemokine MIP-1α, and CCR5 for both chemokines MIP-1α and MIP-1β. In addition, chemokine monocyte chemotactic protein 1 (MCP-1, or CCL2) and its receptor CCR2 are also involved in the recruitment of CD11b + cells in DED. In fact, the corneal infiltrating CD11b + cells in DED have shown significant upregulation of CCR1, CCR2, and CCR5. These infiltrated monocytes and macrophages become activated under the stimulation of environmental TNF-α and IL-1β, and can subsequently produce the same inflammatory cytokines by themselves, thus amplifying the ocular surface inflammation. ,
Another important innate cells called natural killer (NK) cells are also significantly increased in the conjunctiva of mice shortly after desiccating stress, and these NK cells are in activated status by expressing the critical cytokine—interferon-γ (IFN-γ), which not only directly disrupts ocular surface barrier but also promotes corneal APC maturation by upregulating their expressions of MHC-II and costimulators B7—a function bridging adaptive T-cell response in DED. In fact, depletion of NK cells has been shown to lead to decreased levels of cytokines and chemokines responsible for T-cell differentiation and trafficking, thereby resulting in defects in generation and infiltration of activated T cells in DED. ,
Adaptive Immunity Activation
Innate immunity activation causes direct tissue damages as well as facilitates the following antigen-specific, long-lasting adaptive immune response. Essential components bridging the innate and adaptive immunity in DED include NK cells (as described above) and more importantly APC that encompass monocytes and macrophages, dendritic cells, and Langerhans cells. The effectors exerting adaptive immunity comprise of T and B lymphocytes, and a substantial of experimental data demonstrate that CD4 + T-cell immunity plays a central pathogenic role in both Sjögren’s syndrome and non-Sjögren’s DED. , , ,
Ocular surface APC activation and migration
Antigen capture and subsequent presentation by APC to naive T cells is an essential initial step to activate T-cell response. Accordingly, depletion of ocular surface APC has been shown to prevent T-cell activation in DED. In normal healthy cornea, the preponderance of APC is in an “immature” status that is critical to maintain ocular surface immune quiescence and contributes to immune privilege of cornea. However, in DED, significantly increased inflammatory cytokines, such as IL-1β and TNF-α in the ocular surface overcome the existing antiinflammatory factors such as TSP-1 and PEDF , and promote resident APC to acquire “mature” phenotypes by upregulating their MHC-II expression, which is required for APC presentation of antigenic epitopes to T cells. This maturing process is accompanied by the presumed “autoantigen” uptake and processing. To date, the nature of autoantigen(s) in DED remains unclear although kallikrein-13 has been proposed as a putative autoantigen based on its reactivity with sera from Sjögren’s syndrome mice (IQI/Jic mice). The study showed kallikrein-13 expression in multiple tissues where there was coincident lymphocyte infiltration in this transgenic mouse model, including the lacrimal glands, however, the ocular surface tissue was not examined. Further, immunization of rats with a kallikrein family protein led to marked lymphocytic infiltration of the lacrimal glands. In desiccating stress-induced DED mice, increased serum antibodies against kallikrein-13 were detected, and ocular surface tissue expression of kallikrein-13 was reported. Further studies are required to elucidate the endogenous ocular surface antigen(s) that is hypothesized to be exposed upon desiccating stress and provides “danger signals” to resident APC.
To present antigen to and activate naive T cells which primarily reside in secondary draining lymphoid tissues, mature, antigen-bearing APC in ocular surface have to acquire the ability to travel from ocular surface to draining lymph nodes of the eye. This trafficking process once again is tightly regulated by the specific chemokine-receptor axis. Corneal mature MHC-II + APC in DED show significantly enhanced expression of CCR7, a chemokine receptor guiding APC egress toward the draining lymph nodes through enriched environmental CCR7 ligands—CCL19 and CCL21. , Topical blockade of CCR7 has been shown to sufficiently prevent the migration of corneal APC to draining lymph nodes and subsequent T-cell activation in DED. In addition to chemokines, ocular surface APC gain access to lymphoid compartment via newly-formed afferent lymphatic vessels which are lack in normal uninflamed cornea. Interestingly, there is considerable and exclusive growth of lymphatic, but not blood, vessels in DED cornea, that starts early after DED induction from peripheral cornea and advances into central cornea as disease progresses. The selective corneal lymphangiogenesis in DED is dependent on lymphangiogenic-specific vascular endothelial growth factor-C and D (VEGF-C and VEGF-D) and their receptor VEGFR-3. Colocalization of CCR7 + cells with CCL19/21 within corneal lymphatic vessels has been demonstrated in DED cornea.
T-cell activation and expansion in local draining lymph nodes
The central role of adaptive immunity in DED pathogenesis was first demonstrated in the middle of 2000s by an elegant study from the Pflugfelder–Stern–Niederkorn collaboration showing that DED can be induced in healthy mice by adoptive transfer of CD4 + T lymphocytes derived from dry eye mice. In addition, topical cyclosporine-A (CsA), a selective T cell immunosuppressive agent, is one of the first FDA-approved medications to treat DED, providing a strong support for T cells as principal effectors in DED pathogenesis. Recent animal studies have shed considerable light on the precise mechanisms by which how and what subsets of T cells are activated and cause DED via coordinating with an array of other cellular and molecular immune factors. These preclinical studies have significantly facilitated the development of novel therapeutic strategies that are more targeted with less side effects than topical CsA.
Differentiation and activation of naive T cells and expansion of effector T cells
Local draining lymph nodes are the primary site of T-cell activation in DED. Mature migratory CCR7 + APC in local draining lymph nodes express high levels of MHC-II which provides antigenic peptide to cognate naive T cells, as well as B7 (CD80 and CD86) and CD40 costimulatory molecules, which function together with antigen to stimulate T cells. , , Thus, these APC are efficient in priming T cells by upregulating activation markers of CD69 and CD154 (CD40 ligand, CD40L) on T cells (predominantly on CD4 + T subset). Once the immune synapse between APC and T cells is formed, the environmental cytokines, primarily secreted by APC, are predominant factors determining the differentiation fate of primed CD4 + T cells. It is known that the ligation of CD154 with CD40 on APC promotes the production of IL-12 by APC, and increased levels of IL-12 in the milieu of draining lymph nodes of DED mice have been shown. Accordingly, CD4 + T cells show upregulation of IL-12 receptor (IL-12R) expression, and signaling of IL-12/IL-12R promotes naive CD4 + T to differentiate to IFN-γ-expressing Th1 cells, supported by increased levels of the Th1 signature cytokine IFN-γ in DED lymph nodes and conjunctiva, , , despite no confirmation of the cellular source of IFN-γ. Subsequently, the concept of classical Th1 dominance in DED was revised with the characterization of a newly defined CD4 + T-cell subset—IL-17-expressing Th17 cells in DED. , , Initial differentiation of naive T cells toward Th17 lineage is driven by the proinflammatory cytokine IL-6 in the presence of TGF-β, , and increased expression of IL-6 in DED lymph nodes has been demonstrated. , Subsequently, the heightened levels of IL-2 and IL-23 in the lymphoid compartment , further promotes differentiated Th17 cells to proliferate and become activated to gain full function. Unlike most T-cell differentiating and activating cytokines that are mainly produced by APC, the heightened IL-2 is principally from activated effector T cells themselves and acts in an autocrine fashion. In DED, mature APC have been suggested to be the major microenvironmental source of IL-6, TGF-β, and IL-23. Fully activated Th17 cells are capable of secreting effector cytokines, including the signature IL-17 and others such as granulocyte-macrophage colony-stimulating factor (GM-CSF), rendering them pathogenic in DED. , Interestingly, unlike Th1 subset that is regarded as a terminally differentiated CD4 + T-cell lineage and stays relatively stable, the Th17 cells show significant phenotypic and functional plasticity characterized by their ability of acquiring features of other lineages under stimulation of certain signals in the microenvironment. In DED, two subsets of effector Th17 cells have been identified, including IFN-γ − IL-17 + “single-positive” Th17 and IFN-γ + IL-17 + “double-positive” Th17/1. In fact, quantitative analysis of IFN-γ + IL-17 − CD4 + Th1 cells showed no number changes in DED, , suggesting that simple link of increased IFN-γ levels in DED to classic Th1 lineage without examining its cellular source may be inaccurate; instead, the major source of IFN-γ in DED could be the double-positive Th17/1 cells in the disease progression stage while NK cells serve as the primary source in early disease induction phase when adaptive immunity has not been activated. In fact, recent findings have demonstrated that the increased IFN-γ in DED is indeed an integral part of Th17 immunity, as evidenced by the ability of single-positive Th17 cells to convert into double-positive Th17/1 cells with stimulation of environmental IL-12 and IL-23 in DED. Furthermore, double-positive Th17/1 cells are more pathogenic than single-positive Th17 cells, and are required to induce severe acute ocular surface inflammation in DED; in contrast, the classic IL-17 − IFN-γ + Th1 cells isolated from DED are unable to transfer the disease phenotype to normal animals. Taken together, effector Th17 response characterized by their dynamic and coordinated production of various cytokines including IL-17, IFN-γ, and GM-CSF plays a major pathogenic role in DED.
Modulation of effector T-cell activation by regulatory T cells
The dominant players on the immunoregulatory arm that restricts and suppresses excessive inflammation are specialized CD4 + regulatory T cells (Treg), characterized by high expression of CD25 (the high affinity receptor subunit for IL-2 to maintain survival and proliferation of Treg) and Foxp3 (the master transcriptional factor for the development and function of Treg), which are in contrast to proinflammatory CD4 + effector T-cell subsets Th1, Th2, or Th17. To date, two types of Treg have been defined, including the larger population that is developed during the normal process of T-cell maturation in the thymus (termed “natural Treg,” nTreg or tTreg) and the smaller but more specific population that is induced in the periphery from naive T cells in the presence of IL-2 and TGF-β or after encounters with foreign antigens (termed “induced Treg,” iTreg or pTreg). , Both subsets of Treg work in synchrony to maintain peripheral tolerance and immune homeostasis through dampening naive T cell priming or attenuating effector T-cell function. , In DED the regulatory function of Treg is compromised demonstrated by the inability of Treg to suppress IL-17 or IFN-γ production by effector CD4 + T cells despite that the number of Treg is minimally affected. , Furthermore, complete depletion of Treg using an anti-CD25 antibody led to exacerbated DED while reconstitution of normal Treg in mice conferred the recipients resistance to disease induction, suggesting that Treg, probably nTreg, play important regulatory roles in DED pathogenesis. The characteristic phenotype of dysfunctional Treg is downregulation of Foxp3 expression, which has been demonstrated in DED mice. It remained to be elucidated whether other critical Treg function-associated molecules are altered in DED, including surface expression of cell contact-dependent coinhibitory molecules CTLA-4 and GITR as well as secreted antiinflammatory cytokines IL-10 and TGF-β by Treg. It is known that TGF-β is required for the generation of both Treg and Th17, and the presence or absence of the other cytokine IL-6 is a key determinant skewing the immune balance between the two reciprocally interconnected and functionally opposed cell subsets. , As increased IL-6 levels have been consistently observed in both ocular surface and draining lymphoid tissue in DED, , , , , it is plausible to attribute IL-6 as one of the critical factors leading to Treg dysfunction while promoting Th17 in DED. In addition, in vivo blockade of IL-17 effectively restores Treg function in DED, suggesting that IL-17 may also directly or indirectly contributes to Treg dysfunction. Nevertheless, the detailed mechanisms underlying Treg dysfunction in DED, especially their interaction with Th17-associated factors, have yet to be investigated.
CD8 + regulatory T cells have also been implicated in DED pathogenesis by a study demonstrating that depletion of CD8 + T cells in mice promoted the generation of Th17 response and led to a more severe ocular surface disease. The precise subset is assumed to be CD8 + CD103 + cells, which are presumably to limit the Th17 immunity in DED via suppressing the process of APC-mediated Th17 generation, but not restricting already activated Th17 cells.
Ocular surface homing of effector T cells
In addition to the acquisition of the ability to producing inflammatory cytokines, activated effector T cells in the draining lymphoid compartment have to upregulate specific chemokine receptors, thus facilitating their interaction with blood vessel endothelium and their migration to peripheral targeting tissues. In DED, increased conjunctival infiltration of CD3 + T cells has been demonstrated. , These effector CD4 + T cells in DED preferentially upregulate expressions of CCR5, CXCR3, and CCR6, , , directing them to migrate toward ocular surface where higher levels of corresponding chemokines are present, including CCR5 ligands MIP-1α (CCL3), MIP-1β (CCL4), and regulated on activation, normal T-cell expressed and secreted (RANTES, or CCL5); CXCR3 ligands monokine induced by interferon-γ (MIG, or CXCL9), interferon-γ inducible protein 10 (IP-10, or CXCL10); and CCR6 ligand CCL20. , , Among these various T-cell–associated chemokine/receptor axis, CCR6 is discretely expressed by Th17 and CCR6/CCL20 axis has been shown functionally critical for the homing of effector Th17 cells to the ocular surface from the lymphoid compartment, demonstrated by significant abolishment of T-cell infiltration in conjunctiva in the absence of CCR6 (knockout) or after topical neutralization of CCL20 in DED mice. , CCR2, a receptor expressed by monocytes, is also expressed by Th17 cells, particularly on the highly pathogenic IFN-γ + GM-CSF + Th17 subset, and thus CCR2 also contributes to Th17-cell trafficking in DED as demonstrated by decreased T-cell infiltration in conjunctiva after topical blockade of CCR2 in DED mice. In addition to the requirement of guidance by chemokine-receptor axis, peripheralization of T cells needs additional expression of adhesion molecules, such as integrins, which facilitate their binding to extracellular matrix components. In DED, two important pairs including lymphocyte function–associated antigen-1(LFA-1, or integrin α L β 2 ) and its binding ligand intercellular adhesion molecule-1 (ICAM-1), as well as very late antigen-4 (VLA-4, integrin α 4 β 1 ) and its binding ligands such as vascular cell adhesion molecule-1 (VCAM-1) have been demonstrated to mediate T-cell journey to the ocular surface. Specifically, topical application of LFA-1 or VLA-4 small molecule antagonists to DED mice led to significant inhibition of T-cell response in ocular surface. , Lifitegrast, a topical LFA-1 antagonist, has been recently approved by FDA for DED therapy.
Effector T-cell–mediated ocular surface damage
It is widely documented that there are significant infiltration of both IFN-γ − IL-17 + “single-positive” Th17 and IFN-γ + IL-17 + “double-positive” Th17/1, but not IFN-γ + IL-17 − Th1, in DED conjunctiva, , as well as consistently enhanced expressions of IFN-γ and IL-17 in ocular surface of DED. , , , A series of elegant adoptive transfer experiments have further undoubtedly demonstrated that both Th17 and Th17/1 cells are pathogenic effectors in DED with Th17/1 capable of causing more severe tissue destructions due to their ability to additionally secrete IFN-γ. Although lack of new blood vessel formation in DED cornea may limit the direct access of effector T cells to the cornea, cytokines secreted by these cells in the adjacent conjunctiva can diffuse throughout the ocular surface and thus play a major pathogenic role in DED. In fact, IL-17 has been shown to promote corneal production of both MMP-9 and MMP-3 by binding to its receptor IL-17RA that is constitutively expressed by ocular surface epithelia, resulting in corneal epithelial barrier disruption. In addition, IL-17 efficiently induces endothelial and epithelial tissues to secret IL-6, TNF-α, IL-1β, and IL-8, , thus potentially inducing a cytokine cascade at the ocular surface that can propagate tissue damage, further promoting epitope spreading to a more diverse set of target antigens. Furthermore, IL-17 is critical in promoting corneal lymphangiogenesis via inducing expression of prolymphangiogenic VEGF-D by corneal epithelial cells in DED, thus driving progressive ingrowth of lymphatic vessels that allows continuous trafficking of corneal APC to lymphoid compartment and promotes a vicious cycle of autoimmune response. The other pathogenic T-cell cytokine IFN-γ can cause significant conjunctival epithelial squamous metaplasia characterized by loss of goblet cells and decrease of mucin production, accompanied by epithelial apoptosis. , Goblet cells are not only the primary source of mucins for tear film that contributes to the physical barrier but also can directly induce immune tolerance via conditioning APC, and thus loss of conjunctival goblet cells may further exacerbate ocular surface inflammation in DED. Additional fundamental evidence establishing the pathogenic functions of IL-17 and IFN-γ in DED is from preclinical therapeutic tests. In vivo blockade of either IL-17 or IFN-γ leads to significant improvement of corneal epitheliopathy demonstrated by decreased CFS, improved pathological changes, and reduced ocular expression of inflammatory factors. , , , , Besides IL-17 and IFN-γ, Th17-produced GM-CSF also contributes to ocular surface inflammation in DED primarily through promoting ocular surface APC maturation and migration.
Transition of effector T cells to memory T cells and development of chronic inflammation
Clinical DED often presents as a chronic disease, while a majority of experimental data from wild-type animal models reflect an acute inflammatory process; and thus a major gap between experimental models and clinical setting has been identified. Most recent studies accomplished by Dana group have aimed to address this critical question. Frist, they found that when acute DED mice induced by a period of desiccation was transferred to a normal-humidity environment, corneal epitheliopathy in mice gradually regressed to lower levels, but never normalized, for many months, despite absence of tear deficiency, thus establishing a critical murine model of chronic DED. Significant resolution of acute corneal epitheliopathy was seen in the first week of deprivation of desiccation, during which period the previously expanded effector Th17 pool in acute stage dramatically contracted, accompanied by the emergence of a prominent memory Th17 population (CD4 + CD44 high CD62L − IL-17 + ). Memory T-cell–mediated “immunological memory” is a distinct feature of adaptive immunity, and has been shown to play critical pathogenic roles in autoimmunity and chronic inflammation. , Further, it is revealed that these emerging memory Th17 cells during the contraction phase are generated from a small fraction of surviving effector Th17 cells driven by persisting environmental IL-23 stimulation and diminished IL-2 signaling. These memory Th17 cells are functionally pathogenic demonstrated by their ability of transferring DED phenotypes to healthy naive mice. Subsequent work shows that the major effector precursors of memory Th17 cells are double-positive effector Th17/1 cells with single-positive effector Th17 cells contributing relatively less to the memory pool. Along with the chronic low-grade corneal epitheliopathy, memory Th17 cells persist for prolonged period in both conjunctiva and draining lymphoid tissues, and they do not produce IFN-γ. Consistently, only heightened levels of IL-17, but not IFN-γ are expressed in the ocular surface in chronic DED. , In vivo topical blockade of IL-17 in chronic DED can significantly improve the disease severity, demonstrating IL-17 as the principal cytokine sustaining chronicity of DED. The next question is why memory Th17 cells are long-lived in contrast to the short-lived effector Th17 cells. In this regard, two cytokines IL-7 and IL-15 and the distinct expression of their receptors by memory Th17 cells are revealed to be the critical surviving factors for memory Th17 cells via maintaining their robust proliferative and antiapoptotic capabilities. Reactivation of memory Th17 cells by a second desiccating stress on chronic DED mice leads to the development of a more rapid and severe disease exacerbation, suggesting that memory Th17-cell–mediated recall response is responsible for the acute-on-chronic disease exacerbation, akin to intermittent disease “flare” in clinical patients. Blockade of either IL-7 or IL-15 efficiently abolishes memory Th17 cells and the subsequent recall response, preventing the disease flare in chronic DED.
B-cell immunity
B cell is the other major cellular component of adaptive immunity, and B-cell–mediated humoral immunity may participate in disease pathogenicity by primarily differentiating to plasma cells that secret autoantibodies causing self-tissue damages. Autoreactive B cells are known crucial effectors mediating Sjögren’s syndrome evidenced by the presence of autoantibodies directed to ribonuclear proteins SS-A/Ro and SS-B/La in both patients and TSP-1 KO Sjögren’s syndrome mice. , In NOD mouse model of Sjögren’s syndrome, antinuclear antibody (ANA) and type 3 muscarinic acetylcholine receptor (mAChR) autoantibodies (anti-M 3 R) are present in the sera, , which has also been identified as a marker for Sjögren’s syndrome–DED patients. The mAChR is responsible for glandular secretion, and thus the anti-M 3 R autoantibodies are suggested to contribute to the glandular dysfunction in Sjögren’s syndrome together with the glandular infiltrating effector T cells. In fact, immunization of mice with M 3 R peptides led to increased serum levels of anti-M 3 R antibodies and low saliva volume, accompanied by CD4 + T-cell and B-cell infiltrates in the salivary glands. The precise functional relevance of anti-M 3 R antibodies in lacrimal gland dysfunction in Sjögren’s syndrome remains to be determined. In addition, detection of autoantibodies against kallikrein-13 in sera from IQI/Jic mice also suggests potential roles of B-cell immunity in Sjögren’s syndrome–DED. In Aire -deficient mouse model of Sjögren’s syndrome, autoantibody against odorant binding protein 1a (OBP1a) within lacrimal glands is present in the sera, in association with a large proportion of B cell infiltrates in the glands. However, with individual depletion of CD4 + T cells, CD8 + T cells, or B cells, only CD4 + T-cell–depleted group showed significant improvement of lacrimal gland pathology, and further those CD4 + T cells were capable of recognizing OBP1a, suggesting that B-cell immunity in this model plays a limited pathogenic role. In contrast, in Id3 knockout mouse model of Sjögren’s syndrome, depletion of B cells led to disease improvement.
In non-Sjögren’s DED, increased serum antibodies against kallikrein-13 was also detected in mice that were subjected to a long period (3 weeks) of desiccating stress, suggesting that humoral immunity may participate in the pathogenesis of DED subsequent to the initiation of cell-mediated immunity (T-cell immunity). Adoptive transfer of serum or purified IgG collected from the desiccating stress-induced DED mice to T-cell–deficient nude mice led to decreased tear production along with increased cytokines IL-1β, TNF-α, IFN-γ, and IL-17 in tears, as well as reduced conjunctival goblet cells, indicating that autoantibodies contribute to T-cell–dominant pathogenesis in non-Sjögren’s DED. T-cell–dependent B-cell activation is further demonstrated by a recent study showing that CD4 + T cells isolated from desiccation-induced DED mice promote B-cell proliferation and activation primarily via secreted IL-17, and activated B cells further upregulate expression of IL-17 receptor, through which IL-17 further induces B-cell class switching and differentiation to plasma cells.
In addition to their major function of producing autoantibodies in autoimmunity, B cells may functionally serve as APC and secrete various inflammatory cytokines that facilitate T-cell immunity; these areas have yet to be explored in DED pathogenesis.
Lymphocytic inflammation in lacrimal gland
Lacrimal gland, proposed as an integral part of Lacrimal Functional Unit (LFU) along with the ocular surface, can be another target organ of immune attack in DED, supported by data primarily generated from Sjögren’s syndrome–DED models. In both NOD and MRL/lpr mice, significant focal inflammatory infiltrates in lacrimal glands have been shown, and the infiltrating cells consist of dominant CD3 + CD4 + T cells along with small numbers of CD8 + T cells and B220 + B cells, with all of them expressing CD69 activation marker. In addition, there is progressive increase of inflammatory cytokines IL-1β, TNF-α, IL-2, IL-6, IFN-γ, and IL-12, as well as chemokines CCL5, CXCL10, and adhesion molecule ICAM-1 in the lacrimal glands in these genetically modified mice. , , , Blockade of either ICAM-1 or LFA-1 with antibodies can significantly reduce glandular inflammation. These findings suggest a pathogenic role of Th1 response in destroying lacrimal glands. In CD25 KO mice, there are more CD8 + than CD4 + T-cell infiltrates in the lacrimal glands with increased expression of both Th1 (IFN-γ and IL-12) and Th17 (IL-17 and CCL20) cytokines, accompanied by ductal epithelial apoptosis. In TSP-1 KO mice, significant infiltration of CD4 + T cells in the lacrimal glands is noted, and they are active IL-17-secreting Th17 cells, which may further promote the release of inflammatory cytokines IL-1β, TNF-α, and IL-6 by glandular cells. Essential role of Th17 in Sjögren’s syndrome is further illustrated by the ability of IL-17 to promote autoreactive B-cell response and autoantibodies production in NOD mice. Experimental data focusing on salivary glands pathology in Sjögren’s syndrome suggest that T-cell response is critical in inducing disease development, while B-cell response gets involved later in maintaining chronic inflammation, , however, further studies are required to dissect the temporal T- and B-cell responses, as well as differential contributions of Th17 versus Th1, including whether the increased IFN-γ is part of plastic Th17 response, to lacrimal gland destruction in Sjögren’s syndrome–DED. To date, data on lacrimal gland inflammation in non-Sjögren’s DED mice are very limited. A recent study showed in desiccation-induced DED mice increased infiltration of CD11c + dendritic cells in lacrimal glands with activated morphology features observed with a series of intravital multiphoton microscopy in live animals, however, the precise functions of these professional APC in lacrimal gland inflammation remain to be explored.
Disruption of Neuroimmune Homeostasis
Cornea is the most densely innervated tissue in the body, and corneal sensory signals controlled by the trigeminal ganglion neurons are critical in maintaining the ocular surface homeostasis via regulating lacrimation, secretion of epitheliotropic factors, and the blink reflex. Corneal degenerative neuropathy is a frequently encountered pathology in DED, associated with the ocular surface inflammation. In DED induced with either desiccating stress or lacrimal gland excision, acute inflammation of overexpressed inflammatory cytokines IL-1β, IL-6, and TNF-α, sensitizes various nociceptors expressed by sensory nerve terminals at the ocular surface, increases their activity and excitation, and evokes nociceptive pain , resulting from increased activation and discharges of nerves and subsequent release of substance P (SP)—the principal pain mediator. In addition, central sensitization of trigeminal neurons is demonstrated involved in ocular pain sensation in a lacrimal gland excision model. However, with inflammation progression or in severe inflammatory cases, structural damage of corneal nerves occurs evidenced by reduced intraepithelial nerve terminals in the epithelium as well as the decrease of their originating subbasal nerves in both Sjögren’s and non-Sjögren’s DED animals, , , along with the reduced corneal sensitivity or hypoalgesia. , , , Topical blockade of IL-1β with an interleukin-1 receptor antagonist (IL-1Ra) effectively prevents corneal nerve degeneration in DED ( Fig. 4.3 ). On the other hand, altered gene expression and disturbed regeneration of corneal nerves subsequent to acute nerve damage, along with memory Th17-mediated chronic inflammation, may lead to persistent sensory hypersensitivity, thus potentially contributing to chronic pain or discomfort in DED; however, the exact process including both peripheral and central mechanisms remains to be resolved.
