The Cornea in Keratoconjunctivitis Sicca

Abstract

The lacrimal functional unit (LFU) regulates tear production, composition, distribution and clearance to maintain a stable protective tear layer that is essential for maintaining corneal epithelial health. Dysfunction of the LFU, commonly referred to as dry eye, leads to increases tear osmolarity and levels of inflammatory mediators in tears that cause ocular surface epithelial disease, termed keratoconjunctivitis sicca (KCS). Corneal changes in KCS include glycocalyx loss, barrier disruption, surface irregularity inflammatory cytokine/chemokine production, cornification and apoptosis. These can reduce visual function and the increased shear force on the corneal epithelium can stimulate nociceptors sensitized by inflammation causing irritation and pain that may precede frank clinical signs. Therapy of keratoconjunctivitis sicca should be tailored to improve tear stability, normalize tear composition, improve barrier function and minimize shear forces and damaging inflammation to improve corneal epithelial health.

1. Introduction

Dry eye is one of the most prevalent eye conditions, affecting greater than 10 million people in the US alone.(Farrand et al., 2017) Dry eye conditions can be broadly classified into aqueous deficient due to lacrimal hyposecretion leading to reduced tear production/volume and aqueous sufficient (Meibomian gland disease or altered distribution). Dry eye and accompanying tear composition changes cause ocular surface epithelial disease, termed keratoconjunctivitis sicca (KCS). While conjunctival disease decreases secretion of tear stabilizing mucins, corneal epithelial disease can impact visual quality, lead to stromal haze and cause pain.(Khimani et al., 2020; Pflugfelder, 2011) Tear dysfunction is among the most common causes of corneal epithelial disease (Bron et al., 2017)

Maintenance of a supportive precorneal environment is paramount for normal vision. The tear film and cornea are responsible for the majority of light refraction which is fine tuned by lenticular accommodation to focus on the retina. The chronic inflammation of tear dysfunction can compromise corneal clarity and cause as pain. It has been well established that dry eye is an immune based inflammatory disease of the tear secreting and distribution apparatus, termed the Lacrimal Functional Unit (LFU) that results in altered tear composition.(Stern et al., 2004) Normal tears provide several essential functions for the cornea: 1) a lubricative barrier, 2) protection from microbial infection and 3) delivery of trophic and anti-inflammatory factors. The chronic immune based inflammation of dry eye can compromise the cornea protective activity of the three major tear components (lipids, mucins, aqueous). As the most densely innervated surface in the body (40–60x the density of tooth pulp), the cornea is uniquely susceptible to neural sensitization and nociceptive pain induced by inflammatory mediators in dry eye. (Belmonte et al., 2017; Rózsa and Beuerman, 1982)

From a pathological perspective, the cornea inflammation in dry eye is amplified by generation of autoantigens that are phagocytized and processed by activated antigen presenting cells that migrate from the cornea to the regional nodes where they prime and target pathogenic T-cells that home to the LFU. (Saban, 2014; Schaumburg et al., 2011)

2. Methods

A literature search of clinical and basic studies, and review articles published from 1960 to 2020 was performed in PubMed.gov using major terms keratoconjunctivitis sicca, punctate keratopathy, cornea epithelium, tear film, and subheadings glycocalyx, tear osmolarity, matrix metalloproteinases, cytokines, tight junctions. The bibliographies of references identified by this strategy were also reviewed. Attention was given to hypothesis-based studies with replicate data that is statistically valid.

3. The Lacrimal Functional Unit and the Cornea

The LFU is reflexively responds to environmental challenges, such as desiccation, foreign bodies, trauma and surgery to regulate tear secretion and maintain tear composition within its tightly controlled parameters in order to maintain a supportive environment for the ocular surface and in particular to preserve corneal smoothness and clarity.(Stern et al., 2004) Failure to accomplish this in the presence of chronic ocular surface inflammation in dry eye leads to the corneal epithelial disease and pain characteristic of keratitis sicca.(Stern and Pflugfelder, 2017)

Tear flow is initiated within the LFU through sub-conscious stimulation of the dense network of free nerve endings within the corneal epithelium. Studies performed by Belmonte et al. have identified different classes of nociceptors in the cornea and TRPM8+ cold receptors were found to be responsible for stimulating basal tear flow. (Belmonte et al., 2004; Belmonte et al., 2017) (Parra et al., 2010) Polymodal nociceptors have been found to be primary responsible for reflex tear secretion from corneal stimulation.(Acosta et al., 2004) Afferent nerve traffic through the ophthalmic branch of the fifth (Trigeminal) cranial nerve terminate in the pons of the central nervous system where their signals are integrated with autonomic and cortical input. Efferent nerve traffic through the seventh cranial nerve (Facial Nerve) stimulates production of tear components by the lipid secreting meibomian glands in the eyelids, conjunctival goblet cells and the main and accessory lacrimal glands, as well as blinking to distribute them. (Stern et al., 2004) The conjunctiva covers over ninety percent of the ocular surface and secretions from the conjunctival goblet cells contribute to stability of the precorneal tear film. (Alam et al., 2020; Watsky et al., 1988)

The LFU reflex is impacted by internal and external factors that challenge its ability to tightly control tear composition. A person’s hormonal status is one key factor. Androgens are sex steroid hormones that have been reported to suppress inflammation in mouse strains that develop Sjögren syndrome like disease.(Morthen et al., 2019; Rocha et al., 1998) Additionally, dihydrotestosterone suppressed expression of inflammation associated genes and increased anti-inflammatory gene expression in cultured human meibomian gland cells.(Sahin et al., 2020) Androgen levels fall below the protective threshold in peri and post-menopausal women, as well as men on anti-androgen therapy for prostate cancer rendering them susceptible to LFU inflammation. Additionally, people with systemic autoimmune diseases, such as Sjögren syndrome typically have loss of the ability to reflex tear and more severe KCS.(Pflugfelder et al., 1998; Tsubota, 1998; Tsubota et al., 1996) The LFU is compromised in neurotrophic keratitis which exacerbates corneal disease via loss of reflex tearing, blinking and surface desiccation. (Bonini et al., 2003)

4. Homeostatic Maintenance of Corneal Epithelial Smoothness and Barrier Function

A stable tear film and healthy corneal epithelium are required to maintain corneal smoothness, clarity and barrier function that are essential for comfort and high-resolution vision. Evidence indicates the precorneal tear film consists of a lipid layer overlying a secretory mucus/aqueous layer that contains fluid and proteins secreted by the lacrimal glands and MUC5AC mucin secreted by the conjunctival goblet cells that adheres to membrane-associated mucins (MUC1, MUC4, MUC16) in the glycocalyx of the apical epithelium.(Alam et al., 2020; Yokoi et al., 2014) The corneal epithelial barrier is maintained by the glycocalyx and tight junctions in the apical layers of the stratified corneal epithelium that minimize passage of fluid, solutes, inflammatory mediators/cells, and pathogens into the cornea (Figure 1A).(Leong and Tong, 2015) The membrane tethered mucins of the glycocalyx maintain hydrophilicity of the superficial epithelial cells, minimize blink friction and prevent bacterial adherence with MUC16 having a dominant role in maintaining the transcellular barrier.(Uchino, 2018) Silencing of MUC16 in cultured epithelium increased rose bengal dye staining and bacterial adherence, whereas this was not seen with MUC1 knockdown.(Gipson et al., 2014) The tight junction proteins, occludin and zonula occludins (ZO-1), located in the apical corneal epithelial layers maintain the paracellular barrier. (Leong and Tong, 2015) (Chuang et al., 2008; De Paiva et al., 2006a)

Figure 1A.

In normal eyes with a stable tear film, paracellular corneal epithelial barrier is maintained by tight junctions between the apical epithelial cells. Transcellular barrier is provided by the tear film and glycocalyx composed of membrane associated mucins (MAM), particularly MUC16. 1B. In keratoconjunctivitis sicca, osmotic stress from increased sodium (Na+) ions in the tears is a danger signal that activates the JNK and NFκB stress signaling pathways, leading to increased expression of inflammatory cytokines, chemokines and matrix metalloproteinases, in particular MMP-9 that can lyse the tight junction protein, occludin. Epithelial barrier is compromised by loss of glycocalyx and tight junction disruption. Additionally, JNK2 activation stimulates expression of cornified envelope precursors (CEP) that are found in the cornified envelope of the epidermis and may promote apoptosis. Osmotic stress and the reactive oxygen species (ROS) it induces promote NLRP3 inflammasome activation leading to caspase 1 activation and conversion of pro-IL-1β and pro-IL-18 to their active forms.

Keratitis sicca causes corneal surface irregularity that decreases optical contrast sensitivity and increases optical aberrations and light scattering. (Chotikavanich et al., 2009; Goto et al., 2002; Kaido et al., 2011; Rolando et al., 1998) (Liu et al., 2010; Szczotka-Flynn et al., 2019).(Bron et al., 2017; Diaz-Valle et al., 2012) Topographic cornea surface irregularity indices have been found to show significant correlation with the severity of keratitis sicca measured by fluorescein staining. (Liu and Pflugfelder, 1999; Wilson and Klyce, 1991) (de Paiva et al., 2003) (Gumus et al., 2011a; Kojima et al., 2004).

5. Desiccation and Changes in Tear Composition cause Inflammation, Barrier Disruption and Altered Differentiation in the Corneal Epithelium

Disease or dysfunction of the LFU can reduced tear volume and cause changes in tear composition, including elevated osmolarity and concentrations of inflammatory mediators. Increased activity of lipases and proteases can directly destabilize the tears via cleavage of lipids and proteins/glycoproteins, respectively.(Knop and Knop, 2009; Pflugfelder et al., 2005b) Additionally, osmotic stress and inflammatory mediators in the tears can cause death and loss of the glycocalyx-bearing apical corneal epithelium and conjunctival goblet cells that further destabilizes the tears. (Beardsley et al., 2008; Coursey et al., 2016; Deng et al., 2015; Garcia-Posadas et al., 2015; Luo et al., 2007; Yeh et al., 2003) Models have predicted tear osmolarity spikes as high as 900mOsm in areas of precorneal tear film breakup.(Braun et al., 2014; Braun et al., 2015; Peng et al., 2014) Osmotic stress is robust danger signal to the corneal epithelium that activates JNK and NFκB signaling pathways that stimulate expression of cytokines (IL-1β and TNF-α) and chemokines [regulated upon activation normal T cell expressed and secreted (RANTES), monokine induced by interferon-γ (MIG), monocyte chemoattractant protein-1 (MCP-1, CCL-2), and interferon-inducible protein-10 (IP-10)] that are elevated in dry eye tears. (Corrales et al., 2007; Guzman et al., 2015; Guzman et al., 2014; Li et al., 2006; Luo et al., 2005; Na et al., 2012; Pelegrino et al., 2012; Pflugfelder et al., 2005a; Yoon et al., 2007; Zheng et al., 2014) (Figure 1B).

High osmolarity and free radicals induced by osmotic stress activate the NLRP3 inflammasome resulting in activation of Caspase 1 that in turn converts pro-IL-1β and pro-IL-18 to their active forms.(Chi et al., 2017; Deng et al., 2015; Ip and Medzhitov, 2015; Zheng et al., 2014) CCL2, a chemokine elevated in dry eye tears, recruits phagocytic mononuclear cells from the blood that differentiate to macrophages and can be activated by the dry eye environment to produce inflammatory mediators that further amplify corneal inflammation.(Goyal et al., 2009; You et al., 2015) Macrophage and dendritic antigen presenting cells in the cornea and conjunctival can increase MHCII and CCR7 expression, migrate the regional nodes and prime autoreactive CD4+ T cells that can home to the conjunctiva and release cytokines, such as IL-17 that stimulates expression of metalloproteases that disrupt cornea barrier and IFN-γ that can also cause epithelial apoptosis and goblet cell loss.(De Paiva et al., 2009a; De Paiva et al., 2007; Saban, 2014; Schaumburg et al., 2011; Zhang et al., 2014)

The shift to a proinflammatory tear composition in dry eye can cause pathologic changes in the corneal epithelium that characterize the keratitis sicca phenotype. Certain cytokines (IL-8, TNF-α alone, or in combination with IFN-γ) cause shedding of glycocalyx mucin MUC16 from the apical epithelium reducing transcellular barrier. (Albertsmeyer et al., 2010) This is compounded by reduced levels of goblet cell secreted gel forming goblet cell mucin that adheres to the glycocalyx and stabilizes the precorneal tear aqueous/mucin layer.(Khimani et al., 2020) Increased concentration and activity of tear proteases, such as matrix metalloproteinase-9 (MMP-9) can cleave the tight junction protein occludin between apical corneal epithelium that disrupts paracellular barrier.(Pflugfelder et al., 2005b) Accelerated loss, dysfunction or death of apical corneal epithelial cells may also contribute to the corneal barrier disruption in dry eye. (Beardsley et al., 2008; De Paiva et al., 2006a; Yeh et al., 2003) Attrition of the tear film and apical epithelia combined with loss of extracellular matrix results in increased shear and greater mechanical stress on the corneal epithelium and nociceptors.(van Setten, 2020) Similar mechanisms appear to contribute to cornea barrier disruption in other ocular surface inflammatory diseases, including allergy where increased levels of MMP-9 have been detected in the tears of patients with vernal keratoconjunctivitis. (Fukuda and Nishida, 2010; Leonardi et al., 2009)

Cornea barrier disruption is typically detected clinically by fluorescein staining which can show punctate or diffuse staining patterns. Minimal or no punctate fluorescein staining is typically observed in normal corneas; however, fluorescein staining is observed in KCS, is considered a clinical severity marker and is the most commonly used efficacy parameter for clinical trials of dry eye drugs seeking FDA approval. Fluorescein dye staining has been reported to occur by paracellular and transcellular diffusion of dye, with the latter responsible for the punctate pattern.(Bandamwar et al., 2012; Bron et al., 2015) Bandamwar et al. found that healthy cultured corneal epithelial cells showed low level fluorescein staining that increased when stressed with hypertonic saline to induce apoptosis, while dead cells did not stain.(Bandamwar et al., 2014) These findings indicated that punctate fluorescein staining in KCS is due to staining of cells undergoing apoptosis that have a compromised glycocalyx.

Cornea topographic indices can be used to measure smoothness in KCS. The Klyce surface regularity index was found to increase in parallel with the severity of corneal fluorescein staining.(de Paiva et al., 2003; Wilson and Klyce, 1991) Optical coherence tomography (OCT) can be used to measure corneal epithelial thickness. There are conflicting results of studies that evaluated effects of dry eye on corneal epithelial thickness. One study found thinning in superior cornea, but no difference in central and inferior corneal epithelial thickness.(Cui et al., 2014) Another study reported thickening of the corneal epithelium.(Kanellopoulos and Asimellis, 2014) Abou Shousha and colleagues found the corneal epithelium was highly irregular in dry eye due to variability epithelial thickness.(Abou Shousha et al., 2020) Studies evaluating the effects of dry eye on corneal epithelial proliferation have also reported conflicting results. A significantly increased number of cells stained by the proliferation marker KI67 was noted in the corneal epithelium of two different autoimmune prone mutant mouse strains that develop Sjögren syndrome like KCS (CD25−/− and Aire−/−).(Efraim et al., 2020; Stepp et al., 2018b) Increased proliferation was also noted in 5 week old mouse autoimmune prone mouse strain that develops Sjogren syndrome like KCS.(Efraim et al., 2020) In contrast, no difference in the number of proliferating corneal epithelial cells was noted in mice subjected to desiccating stress, compared to unstressed control mice.(Stepp et al., 2018a)

A shift toward a skin like phenotype in the cornea has been found in mouse models of dry eye. Increased expression of cornified envelope precursor proteins, involucrin, and small proline rich peptides (SPRRs) and transglutaminase enzyme 1 (Tg-1) that crosslinks them has been reported in the mouse corneal epithelium after exposure to desiccating stress.(Corrales et al., 2011) This shift in differentiation pattern in the cornea epithelium can be induced by osmotic stress via activation of c-jun N-terminal kinase 2 (JNK2). (Chen et al., 2008; De Paiva et al., 2009b) These changes may contribute to the decreased wettability and increased stiffness of the corneal epithelium in KCS. (van Setten, 2020) Interestingly in the mouse desiccating stress model, we’ve observed that staining of the corneal epithelium with a 70kDa fluorescent dextran molecule decreases as expression of cornified envelope precursors increases, suggesting that the shift towards cornification may be a compensatory change to reestablish an alternative corneal barrier in response to desiccating stress. Indeed, visible cornification of the corneal epithelium may be observed in severe cases of KCS in conditions, such as Stevens Johnson syndrome.(Wall et al., 2003) It is possible there is lower level nonvisible expression of cornifying factors in less severe KCS.

The human sub basal nerve plexus can be imaged by in vivo laser scanning confocal microscopy; however, current instruments lack the sensitivity to detect intraepithelial nerve endings.(Villani et al., 2014) Several studies have reported a decrease in sub basal nerve density in Sjögren syndrome associated KCS.(Benitez del Castillo et al., 2004; Labbe et al., 2013; Villani et al., 2007) Additionally, dendritic cell infiltration around nerves and morphological changes, including bead like formations, nerve sprouting and thickened stromal nerves have been reported.(Labbe et al., 2013; Tuisku et al., 2008) Osmotic stress, inflammation and nerve damage from dry eye sensitize and increase activity in the remaining corneal nociceptors which may be interpreted as dryness and eye pain in KCS.(Belmonte et al., 2017)

Confocal microscopy with βIII tubulin immunostaining of nerve fibers in murine dry eye models has been used to study the time course and effects of dryness on epithelial nerve endings and the subepithelial plexus.(Stepp et al., 2018a) These studies confirm the effects of KCS on corneal nerves that were seen in human images. Axon density in the mouse cornea was found to decrease after 5 and 10 days of exposure to desiccating stress (DS), while the density of intraepithelial nerve fibers was significantly decreased as early as 3 days and continued to decrease up to 10 days of desiccating stress.(Stepp et al., 2018a) This was accompanied by reduced corneal sensitivity as early as 3 days following exposure to DS. Axon density and corneal sensitivity were also decreased in 24-month-old C57BL/6 mice that develop KCS by this age. Decreased expression of several genes that regulate axon growth and elongation was found in the corneal epithelium of the aged mice. (Stepp et al., 2018a) Reduction in cornea sensitivity and axon density was also noted in the CD25−/− strain with autoimmune KCS as early as 4 weeks of age. (Stepp et al., 2018b)

6. Strategies to treat keratitis sicca

Therapy of keratitis sicca should be tailored to address the cellular and molecular disease mechanisms we’ve highlighted. Osmoprotectants, such as L-carnitine, erythritol and trehalose, that are found in artificial tears, have been found to decrease inflammation, metalloproteinase production and oxidative damage, and promote autophagic flux in osmotically stressed corneal epithelial cells. (Deng et al., 2014; Hua et al., 2015; Hua et al., 2014; Liu et al., 2020) Corticosteroids decrease NFκB activation, cytokine and MMP production and have been found to preserve cornea barrier function in response to DS.(De Paiva et al., 2006a; De Paiva et al., 2006b; Moore et al., 2015; Pinto-Fraga et al., 2018; Scudeletti et al., 1996; Solomon et al., 2000) Cyclosporine A inhibits dry eye associated apoptosis in ocular surface epithelia.(Gao et al., 1998; Strong et al., 2005) Both cyclosporine and autologous serum or platelet rich plasma have been reported to increase number of conjunctival goblet cells that secrete tear stabilizing mucin.(Alio et al., 2007; Kunert et al., 2002; Noble et al., 2004; Pflugfelder et al., 2008) Recombinant human nerve growth factor is now approved to treat neurotrophic keratitis. Numerical improvement in corneal nerve density and corneal sensitivity were observed following 8 weeks of this therapy.(Pflugfelder et al., 2020) It is possible that recombinant NGF has the potential to restore corneal innervation in chronic KCS. Furthermore, this therapy highlights the possibility of using other topical biologics in the future to decrease inflammation and restore corneal homeostasis in KCS.

For severe corneal epitheliopathy, scleral lenses have proven to be an excellent option for improving irritation symptoms and visual acuity.(Dimit et al., 2013; Gumus et al., 2011b; Jacobs and Rosenthal, 2007; Romero-Rangel et al., 2000) These devices create a fluid filled reservoir that shields the corneal from mechanical shear forces and disease-causing inflammatory mediators in the tears. They also shield the corneal nociceptors from noxious stimuli and the body temperature saline reservoir minimizes corneal cooling and nerve firing during in the inter blink intervals.

7. Summary

KCS sicca is one of the most common eye conditions and improvement of corneal epithelial disease is the most commonly used endpoint in dry eye therapeutic trials. Irritation and visual disturbance from corneal epithelial disease can greatly impact productivity and quality of life. Changes in tear composition, such as increased osmolarity, activate stress pathways in the corneal epithelium that can directly and indirectly disrupt barrier function and lead to a poorly lubricated, irregular corneal surface. Barrier disruption and sensitization of nociceptors in the cornea is responsible for irritation and pain in KCS. Therapy of the corneal epithelial disease of KCS should include agents that target disease mechanisms.

Highlights.

This review highlights the effects of dry eye on the corneal epithelium. The corneal epithelium has an essential function of protecting the cornea from environmental insults and inflammation. It also shields the nociceptors in the cornea from damage and sensitizing agents. Reduced volume and tear compositional changes in dry eye cause dysfunction and death of the corneal epithelium that alters barrier function and differentiation, and stimulates inflammation. These changes degrade visual function and cause eye dryness and pain sensations.